Methods for treating a disorder by using Mage-3 or Mage-3 related materials

ABSTRACT

The invention relates to nucleic acid molecules which code for the tumor rejection antigen precursor MAGE-3. Also disclosed are vectors, cell lines, and so forth, which utilize the nucleic acid molecule, and optionally, molecules coding for human leukocyte antigen HLA-A1. Uses of these materials in therapeutic and diagnostic contexts are also a part of the invention.

RELATED APPLICATION

This application is a divisional of Ser. No. 08/967727, filed on Nov.12, 1997, now U.S. Pat. No. 6,025474, which is a divisional of Ser. No.08/037230, filed on Mar. 26, 1993, now U.S. Pat. No. 6,235525, which isa continuation-in-part of PCT Application PCT/US92/04354 filed on May22, 1992, designating the United States, which is a continuation-in-partof Ser. No. 07/807043, filed on Dec. 12, 1991, now U.S. Pat. No.5,342774, which is a continuation-in-part of Ser. No. 07/764365, filedon Sep. 23, 1991, now abandoned, which is a continuation-in-part of Ser.No. 07/728838, filed on Jul. 9, 1991, now abandoned, which is acontinuation-in-part of Ser. No. 07/705702, filed on May 23, 1991, nowabandoned.

FIELD OF THE INVENTION

This invention relates in general to the field of immunogenetics asapplied to the study of oncology. More specifically, it relates to thestudy and analysis of mechanisms by which tumors are recognized by theorganism's immune system such as through the presentation of so-calledtumor rejection antigens, and the expression of what will be referred toherein as “tumor rejection antigen precursors” or “TRAPs”. Mostspecifically, it refers to nucleic acid molecules coding for one suchTRAP, i.e., MAGE-3, which is processed to a tumor rejection antigen or“TRA” presented by HLA-A1 molecules.

BACKGROUND AND PRIOR ART

The study of the recognition or lack of recognition of cancer cells by ahost organism has proceeded in many different directions. Understandingof the field presumes some understanding of both basic immunology andoncology.

Early research on mouse tumors revealed that these displayed moleculeswhich led to rejection of tumor cells when transplanted into syngeneicanimals. These molecules are “recognized” by T-cells in the recipientanimal, and provoke a cytolytic T-cell response with lysis of thetransplanted cells. This evidence was first obtained with tumors inducedin vitro by chemical carcinogens, such as methylcholanthrene. Theantigens expressed by the tumors and which elicited the T-cell responsewere found to be different for each tumor. See Prehn, et al., J. Natl.Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572(1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30:2458-2462 (1970) for general teachings on inducing tumors with chemicalcarcinogens and differences in cell surface antigens. This class ofantigens has come to be known as “tumor specific transplantationantigens” or “TSTAs”. Following the observation of the presentation ofsuch antigens when induced by chemical carcinogens, similar results wereobtained when tumors were induced in vitro via ultraviolet radiation.See Kripke, J. Natl. Canc. Inst. 53: 333-1336 (1974).

While T-cell mediated immune responses were observed for the types oftumor described supra, spontaneous tumors were thought to be generallynon-immunogenic. These were therefore believed not to present antigenswhich provoked a response to the tumor in the tumor carrying subject.See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).

The family of tum antigen presenting cell lines are immunogenic variantsobtained by mutagenesis of mouse tumor cells or cell lines, as describedby Boon et al., J. Exp. Med. 152: 1184-1193 (1980), the disclosure ofwhich is incorporated by reference. To elaborate, tum⁻ antigens areobtained by mutating tumor cells which do not generate an immuneresponse in syngeneic mice and will form tumors (i.e., “tum⁺” cells).When these tum⁺ cells are mutagenized, they are rejected by syngeneicmice, and fail to form tumors (thus “tum⁻”). See Boon et al., Proc.Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which isincorporated by reference. Many tumor types have been shown to exhibitthis phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).

It appears that tum⁻ variants fail to form progressive tumors becausethey elicit an immune rejection process. The evidence in favor of thishypothesis includes the ability of “tum⁻” variants of tumors, i.e.,those which do not normally form tumors, to do so in mice with immunesystems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl,Acad. Sci. USA 76: 5282-5285 (1979); and the observation thatintraperitoneally injected tum⁻ cells of mastocytoma P815 multiplyexponentially for 12-15 days, and then are eliminated in only a few daysin the midst of an influx of lymphocytes and macrophages (Uyttenhove etal., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes theobservation that mice acquire an immune memory which permits them toresist subsequent challenge to the same tum⁻ variant, even whenimmunosuppressive amounts of radiation are administered with thefollowing challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra).Later research found that when spontaneous tumors were subjected tomutagenesis, immunogenic variants were produced which did generate aresponse. Indeed, these variants were able to elicit an immuneprotective response against the original tumor. See Van Pel et al., J.Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it ispossible to elicit presentation of a so-called “tumor rejection antigen”in a tumor which is a target for a syngeneic rejection response. Similarresults have been obtained when foreign genes have been transfected intospontaneous tumors. See Fearson et al., Cancer Res. 48: 2975-1980 (1988)in this regard.

A class of antigens has been recognized which are presented on thesurface of tumor cells and are recognized by cytotoxic T cells, leadingto lysis. This class of antigens will be referred to as “tumor rejectionantigens” or “TRAs” hereafter. TRAs may or may not elicit antibodyresponses. The extent to which these antigens have been studied, hasbeen via cytolytic T cell characterization studies, in vitro i.e., thestudy of the identification of the antigen by a particular cytolytic Tcell (“CTL” hereafter) subset. The subset proliferates upon recognitionof the presented tumor rejection antigen$ and the cells presenting theantigen are lysed. Characterization studies have identified CTL cloneswhich specifically lyse cells expressing the antigens. Examples of thiswork may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boonet al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol.124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634(1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladinoet al., Canc. Res. 47: 5074-5079 (1987). This type of analysis isrequired for other types of antigens recognized by CTLs, including minorhistocompatibility antigens, the male specific H-Y antigens, and a classof antigens, referred to as “tum−” antigens, and discussed herein.

A tumor exemplary of the subject matter described supra is known asP815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278(1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al.,J. Exp. Med. 172: 35-45 (1990), the disclosures of which areincorporated by reference. The P815 tumor is a mastocytoma, induced in aDBA/2 mouse with methylcholanthrene and cultured as both an in vitrotumor and a cell line. The P815 line has generated many tum⁻ variantsfollowing mutagenesis, including variants referred to as P91A (DePlaen,supra), 35B (Szikora, supra), and P198 (Sibille, supra). In contrast totumor rejection antigens—and this is a key distinction—the tum⁻ antigensare only present after the tumor cells are mutagenized. Tumor rejectionantigens are present on cells of a given tumor without mutagenesis.Hence, with reference to the literature, a cell line can be tum⁺, suchas the line referred to as “P1”, and can be provoked to producetumvariants. Since the tum phenotype differs from that of the parentcell line, one expects a difference in the DNA of tum⁻ cell lines ascompared to their tum⁺ parental lines, and this difference can beexploited to locate the gene of interest in tum⁻ cells. As a result, itwas found that genes of tum⁻ variants such as P91A, 35B and P198 differfrom their normal alleles by point mutations in the coding regions ofthe gene. See Szikora and Sibille, supra, and Lurguin et al., Cell 58:293-303 (1989). This has proved not to be the case with the TRAs of thisinvention. These papers also demonstrated that peptides derived from thetum⁻ antigen are presented by the L^(d) molecule for recognition byCTLs. P91A is presented by L^(d), P35 by D^(d) and P198 by K^(d).

Prior patent applications PCT/US92/04354, U.S. Ser. Nos. 807,043;764,364; 728,838 and 707,702, all of which are incorporated byreference, describe inventions involving, inter alia, genes and othernucleic acid molecules which code for various TRAPs, which are in turnprocessed to tumor rejection antigen, or “TRAs”.

The genes are useful as a source for the isolated and purified tumorrejection antigen precursor and the TRA themselves, either of which canbe used as an agent for treating the cancer for which the antigen is a“marker”, as well as in various diagnostic and surveillance approachesto oncology, discussed infra. It is known, for example, that tum⁻ cellscan be used to generate CTLs which lyse cells presenting different tum⁻antigens as well as tum⁺ cells. See, e.g., Maryanski et al., Eur. J.Immunol 12: 401 (1982); and Van den Eynde et al., Modern Trends inLeukemia IX (June 1990), the disclosures of which are incorporated byreference. The tumor rejection antigen precursor may be expressed incells transfected by the gene, and then used to generate an immuneresponse against a tumor of interest.

In the parallel case of human neoplasms, it has been observed thatautologous mixed lymphocyte-tumor cell cultures (“MLTC” hereafter)frequently generate responder lymphocytes which lyse autologous tumorcells and do not lyse natural killer targets, autologous EBV-transformedB cells, or autologous fibroblasts (see Anichini et al., Immunol. Today8: 385-389 (1987)). This response has been particularly well studied formelanomas, and MLTC have been carried out either with peripheral bloodcells or with tumor infiltrating lymphocytes. Examples of the literaturein this area including Knuth et al., Proc. Natl. Acad. Sci. USA 86:2804-2802 (1984); Mukherji et al., J. Exp. Med. 158: 240 (1983) ; Hérinet all, Int. J. Canc. 39: 390-396 (1987); Topalian et al, J. Clin. Oncol6: 839-853 (1988). Stable cytotoxic T cell clones (“CTLs” hereafter)have been derived from MLTC responder cells, and these clones arespecific for the tumor cells. See Mukherji et al., supra, Herin et all,supra, Knuth et al., supra. The antigens recognized on tumor cells bythese autologous CTLs do not appear to represent a cultural artifact,since they are found on fresh tumor cells. Topalian et al., supra;Degiovanni et al., Eur. J. Immunol. 20: 1865-1868 (1990). Theseobservations, coupled with the techniques used herein to isolate thegenes for specific murine tumor rejection antigen precursors, have ledto the isolation of nucleic acid sequences coding for tumor rejectionantigen precursors of TRAs presented on human tumors. It is now possibleto isolate the nucleic acid sequences which code for tumor rejectionantigen precursors, including, but not being limited to those mostcharacteristic of a particular tumor, with ramifications that aredescribed infra.

Additional work has focused upon the presentation of TRAs by the classof molecules known as human leukocyte antigens, or “HLAs”. This work hasresulted in several unexpected discoveries regarding the field.Specifically in U.S. patent application Ser. No. 938,334, the disclosureof which is incorporated by reference, nonapeptides are taught which arepresented by the HLA-A1 molecule. The reference teaches that given theknown specificity of particular peptides for particular HLA molecules,one should expect a particular peptide to bind one HLA molecule, but notothers. This is important, because different individuals possessdifferent HLA phenotypes. As a result, while identification of aparticular peptide as being a partner for a specific HLA molecule hasdiagnostic and therapeutic ramifications, these are only relevant forindividuals with that particular HLA phenotype. There is a need forfurther work in the area, because cellular abnormalities are notrestricted to one particular HLA phenotype, and targeted therapyrequires some knowledge of the phenotype of the abnormal cells at issue.

In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993 andincorporated by reference, the fact that the MAGE-1 expression productis processed to a second TRA is disclosed. This second TRA is presentedby HLA-C10-molecules. The disclosure shows that a given TRAP can yield aplurality of TRAs.

In U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, andincorporated by reference herein, tyrosinase is described as a tumorrejection antigen precursor. This reference discloses that a moleculewhich is produced by some normal cells (e.g., melanocytes), is processedin tumor cells to yield a tumor rejection antigen that is presented byHLA-A2 molecules.

It was mentioned, supra, that different individuals possess differentHLA types. It has also been found that the expression of particular MAGEgenes is not always linked to particular disorders, or individuals ofparticular HLA types. Thus, one cannot state, e.g., that all melanomapatients will express MAGE-1 TRAP nor could one say categorically thatMAGE-1 expression is limited to melanoma patients of type HLA-A1.Further, one cannot state that only one type of TRAP is expressed inindividuals of a particular HLA type. No rules or guidelines can bepointed to which correlate any of these factors.

Thus, it is not expected that a second TRAP is processed to a TRAP whichis presented by HLA-A1 molecules. It has now been found that in additionto MAGE-1, a TRA derived from MAGE-3 TRAP is presented by HLA-A1molecules. This is shown in examples 37-40, which follow, together witha discussion of the ramifications of this discovery.

These and various other aspects of the invention are elaborated upon inthe disclosure which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts detection of transfectants expressing antigen P815A.

FIG. 2 shows the sensitivity of clones P1.HTR, P0.HTR, genomictransfectant P1A.T2 and cosmid transfectant P1A.TC3.1 to lysis byvarious CTLs, as determined by chromium release assays.

FIG. 3 is a restriction map of cosmid C1A.3.1.

FIG. 4 shows Northern Blot analysis of expression of gene P1A.

FIG. 5 sets forth the structure of gene P1A with its restriction sites.

FIG. 6 shows the results obtained when cells were transfected with thegene from P1A, either isolated from P815 or normal cells and then testedwith CTL lysis.

FIG. 7 shows lytic studies using mast cell line L138. 8A.

FIG. 8 is a map of subfragments of the 2.4 kb antigen E fragmentsequence which also express the antigen.

FIG. 9 shows homology of sections of exon 3 from genes mage 1, 2 and 3.

FIG. 10 shows the result of Northern blots for MAGE genes on varioustissues.

FIG. 11 presents the data of FIG. 13 in table form.

FIG. 12 shows Southern Blot experiments using the various human melanomacell lines employed in this application.

FIG. 13 is a generalized schematic of the expression of MAGE 1, 2 and 3genes by tumor and normal tissues.

FIG. 14 shows results from a chromium release assay using CTL clone20/38 on various cell lines.

FIG. 15 presents the result of assays undertaken to determine antigenicspecificity of CTL clone 20/38.

FIG. 16 shows the results obtained when a TNF release assay was carriedout on various transfected cells.

BRIEF DESCRIPTION OF SEQUENCES

SEQ ID NO: 1 is cDNA for part of gene P1A.

SEQ ID NO: 2 presents coding region of cDNA for gene P1A.

SEQ ID NO: 3 shows non coding DNA for P1A cDNA which is 3′ to the codingregion of SEQ ID NO: 2.

SEQ ID NO: 4 is the entire sequence of cDNA for P1A.

SEQ ID NO: 5 is the genoinic DNA sequence for P1A.

SEQ ID NO: 6 shows the amino acid sequence for the antigenic peptidesfor P1A TRA. The sequence is for cells which are A⁺ B⁺, i.e., expressboth the A and B antigens.

SEQ ID NO: 7 is a nucleic acid sequence coding for antigen E.

SEQ ID NO: 8 is a nucleic acid sequence coding for MAGE-1.

SEQ ID NO: 9 is the gene for MAGE-2.

SEQ ID NO: 10 is the gene for MAGE-21.

SEQ ID NO: 11 is cDNA for MAGE-3.

SEQ ID NO: 12 is the gene for MAGE-31.

SEQ ID NO: 13 is the gene for MAGE-4.

SEQ ID NO: 14 is the gene for MAGE-41.

SEQ ID NO: 15 is cDNA for MAGE-4.

SEQ ID NO: 16 is cDNA for MAGE-5.

SEQ ID NO: 17 is genomic DNA for MAGE-51.

SEQ ID NO: 18 is cDNA for MAGE-6.

SEQ ID NO: 19 is genomic DNA for MAGE-7.

SEQ ID NO: 20 is genomic DNA for MAGE-8.

SEQ ID NO: 21 is genomic DNA for MAGE-9.

SEQ ID NO: 22 is genomic DNA for MAGE-10.

SEQ ID NO: 23 is genomic DNA for MAGE-11.

SEQ ID NO: 24 is genomic DNA for smage-I.

SEQ ID NO: 25 is genomic DNA for smage-II.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Many different “MAGE” genes have been identified, as will be seen fromthe sequences which follow the application. The protocols described inthe following examples were used to isolate these genes and cDNAsequences.

“MAGE” as used herein refers to a nucleic acid sequence isolated fromhuman cells. The acronym “smage” is used to describe sequences of murineorigin.

When “TRAP” or “TRAs” are discussed herein as being specific to a tumortype, this means that the molecule under consideration is associatedwith that type of tumor, although not necessarily to the exclusion ofother tumor types.

EXAMPLE 1

In order to identify and isolate the gene coding for antigen P815A, genetransfection was used. This approach requires both a source of the gene,and a recipient cell line. Highly transfectable cell line P1.HTR was thestarting material for the recipient, but it could not be used withoutfurther treatment, as it presents “antigen A”, one of four recognizedP815 tumor antigens. See Van Pel et al., Molecular Genetics 11: 467-475(1985). Thus, screening experiments were carried out to isolate celllines which did not express the antigen and which nonetheless possessedP1.HTR's desirable qualities.

To do this, P1.HTR was screened with CTLs which were specific for eachof tumor antigens A, B, C and D. Such CTLs are described by Uyttenhoveet al., J. Exp. Med. 157: 1040-1052 (1983).

To carry out the selection, 10⁶ cells of P1.HTR were mixed with 2-4×10⁶cells of the CTL clone in a round bottom tube in 2 ml of medium, andcentrifuged for three minutes at 150×g. After four hours at 37° C., thecells were washed and resuspended in 10 ml of medium, followingMaryanski et al., Eur. J. Immunol. 12: 406-412 (1982). Additionalinformation on the CTL assay and screening protocol, in general may befound in Boon et al., J. Exp. Med. 152: 1184-1193 (1980), and Maryanskiet al., Eur. J. Immunol. 12: 406-412 (1982), the disclosure of which areincorporated by reference.

When these selections were carried out, a cell line variant was foundwhich expressed neither antigen A or B. Additional selections with CTLsspecific for antigen C then yielded a variant which also lacked antigenC. Please see FIG. 2 for a summary of the results of these screenings.The variant PO.HTR is negative for antigens A, B and C, and was:therefore chosen for the transfection experiments.

The cell line PO.HTR has been deposited in accordance with the BudapestTreaty at the Institute Pasteur Collection Nationale De Cultures DeMicroorganismes, 28, Rue de Docteur Roux, 75724 Paris France, and hasaccession number I-1117.

This methodology is adaptable to secure other cell lines which arevariants of a cell type which normally presents at least one of the fourrecognized P815 tumor antigens, i.e., antigens A, B, C and D, where thevariants present none of antigens A, B and C. P1.HTR is a mastocytomacell line, so it will be seen that the protocol enables the isolation ofbiologically pure mastocytoma cell lines which express none of P815antigens A, B and C, but which are highly transfectable. Other tumortypes may also be screened in this fashion to secure desired,biologically pure cell lines. The resulting cell lines should be atleast as transfectable with foreign DNA as is P1.HTR, and should beselected so as to not express a specific antigen.

EXAMPLE 2

Previous work reported by DePlaen et al., Proc. Natl. Acad. Sci. USA 85:2274-2278 (1988) the disclosure of which is incorporated by referenceherein had shown the efficacy of using cosmid library transfection torecover genes coding for tum⁻ antigens.

Selective plasmid and genomic DNA of P1.HTR were prepared, followingWölfel et al., Immunogenetics 26: 178-187 (1987). The transfectionprocedure followed Corsaro et al., Somatic Cell Molec. Genet 7: 603-616(1981), with some modification. Briefly, 60 μg of cellular DNA and 3 μgof DNA of plasmid pHMR272, described by Bernard et al., Exp. Cell. Biol.158: 237-243 (1985) were mixed. This plasmid confers hygromycinresistance upon recipient cells, and therefore provides a convenient wayto screen for transfectants. The mixed DNA was combined with 940 ul of 1mM Tris-HCl (pH 7.5), 0.1 mM EDTA; and 310 ul 1M CaCl₂. The solution wasadded slowly, and under constant agitation to 1.25 ml of 50 mM Hepes,280 mM NaCl, 1.5 mM Na₂HPO₄, adjusted to pH 7.1 with NaOH. Calciumphosphate-DNA precipitates were allowed to form for 30-45 minutes atroom temperature. Following this, fifteen groups of PO.HTR cells (5×10⁶)per group were centrifuged for 10 minutes at 400 g. Supernatants wereremoved, and pellets were resuspended directly into the mediumcontaining the DNA precipitates. This mixture was incubated for 20minutes at 37° C., after which it was added to an 80 cm² tissue cultureflask containing 22.5 ml DMEM, supplemented with 10% fetal calf serum.After 24 hours, medium was replaced. Forty-eight hours aftertransfection, cells were collected and counted. Transfected cells wereselected in mass culture using culture medium supplemented withhygromycin B (350 ug/ml). This treatment selected cells for hygromycinresistance.

For each group, two flasks were prepared, each containing 8×10⁶ cells in40 ml of medium. In order to estimate the number of transfectants, 1×10⁶cells from each group were plated in 5 ml DMEM with 10% fetal calf serum(FCS), 0.4% bactoagar, and 300 ug/ml hygromycin B. The colonies werethen counted 12 days later. Two independent determinations were carriedout and the average taken. This was multiplied by 5 to estimate thenumber of transfectants in the corresponding group. Correction had to bemade for the cloning efficiency of P815 cells, known to be about 0.3.

EXAMPLE 3

Eight days after transfection as described in example 2, supra,antibiotic resistant transfectants were separated from dead cells, usingdensity centrifugation with Ficoll-Paque. These cells were maintained innon-selective medium for 1 or 2 days. The cells were plated in 96 wellmicroplates (round bottom), at 30 cells/microwell in 200 ul of culturemedium. Anywhere from 100-400 microwells were prepared, depending on thenumber of transfectants prepared. Agar colony tests gave estimates of500-3000. After 5 days, the wells contained about 6×10⁴ cells andreplicate plates were prepared by transferring {fraction (1/10)} of thewells to microplates which were then incubated at 30° C. One day later,master plates were centrifuged, medium removed, and 750 CTLs againstP815 antigen A (CTL-P1:5) were added to each well together with 10⁶irradiated syngeneic feeder spleen cells in CTL culture mediumcontaining 40 U/ml recombinant human IL-2, and HAT medium to killstimulator cells. Six days later, plates were examined visually toidentify wells where CTLs had proliferated. Where plates showedproliferating microcultures,aliquots of 100 ul of the wells weretransferred to another plate containing ⁵¹Cr labeled P1.HTR target cells(2×10³-4×10³ per well), and chromium release was measured after 4 hours.Replicate microcultures corresponding to those showing high CTL activitywere expanded and cloned by limited dilution in DMEM with 10% FCS. Fivedays later, about 200 clones were collected and screened with theCTL.P1:5 cell line, described supra, in a visual lysis assay. See FIG.1A for these results.

In these experiments, three of the fifteen groups of transfectantsyielded a few positive microcultures. These microcultures were testedfor lytic activity against P1.HTR, as described supra. Most of themicrocultures where proliferation was observed showed lytic activity.This activity was well above background, as shown in FIG. 1B. Thisfigure summarizes data wherein two groups of cells (groups “5” and“14”), 400 and 300 microwells were seeded with 30 hygromycin resistanttransfected cells. Amplification and duplication of the microcultureswas followed by addition of anti-A CTL P1:5. Six days later, lyticactivity against P1.HTR was tested. In the figure, each point representslytic activity of a single microculture.

Duplicate microcultures corresponding to several positive wells weresubcloned, and more than 1% of the subclones were found to be lysed byanti-A CTL. Thus, three independent transfectants expressing P815A wereobtained from 33,000 hygromycin resistant transfectants. One of theselines, referred to hereafter as P1A.T2 was tested further.

The relevant antigen profile of P1A.T2 is shown in FIG. 2, this beingobtained via anti-CTL assays of the type described supra.

EXAMPLE 4

The CTL assays carried out for P1A.T2 demonstrated that it presentedantigen A (“P815A”), and therefore had received the gene from P1.HTR. Tothat end, this cell line was used as a source for the gene for theantigen precursor in the following experiments.

Prior work had shown that genes coding for tum⁻ antigens could berecovered directly from transfectants obtained with a cosmid library.See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988).This procedure was followed for recovery of the P815 gene.

Total genomic DNA of P1A.T2 was partially digested with restrictionendonuclease Sau 3A1, and fractionated by NaCl density gradientultracentrifugation to enrich for 35-50 kb DNA fragments, followingGrosveld et al., Gene 10: 6715-6732 (1982). These fragments were ligatedto cosmid arms of C2RB, described by Bates et al., Gene 26: 137-146(1983), the disclosure of which is incorporated by reference. Thesecosmid arms had been obtained by cleavage with SmaI and treatment withcalf intestinal phosphatase, followed by digestion with BamHI. LigatedDNA was packaged into lambda phage components, and titrated on E. coliED 8767, following Grosveld et al., supra. Approximately 9×10⁵ampicillin resistant colonies were obtained per microgram of DNA insert.

The cosmid groups were amplified by mixing 30,000 independent cosmidswith 2 ml of ED 8767 in 10 mM MgCl₂, incubated 20 minutes at 37° C.,diluted with 20 ml of Luria Bertani (“LB”) medium, followed byincubation for one hour. This suspension was titrated and used toinoculate 1 liter of LB medium in the presence of ampicillin (50 ug/ml).At a bacterial concentration of 2×10⁸ cells/ml (OD₆₀₀=0.8), a 10 mlaliquot was frozen, and 200 ug/ml chloramphenicol was added to theculture for overnight incubation. Total cosmid DNA was isolated byalkaline lysis procedure, and purified on CsCl gradient.

In these experiments, a library of 650,000 cosmids was prepared. Theamplification protocol involved the use of 21 groups of approximately30,000 cosmids.

EXAMPLE 5

Using the twenty-one groups of cosmids alluded to supra, (60 ug) and 4ug of pHMR272, described supra, groups of 5×10⁶ PO.HTR cells were usedas transfectant hosts. Transfection was carried out in the same manneras described in the preceding experiments. An average of 3000transfectants per group were tested for antigen presentation, againusing CTL assays as described. One group of cosmids repeatedly yieldedpositive transfectants, at a frequency of about {fraction (1/5,000)}drug resistant transfectants. The transfectants, as with P1A.T2, alsoshowed expression of both antigen A and B. The pattern of expression oftransfectant P1A.TC3.1 is shown in FIG. 2.

EXAMPLE 6

As indicated in Example 51 supra, three independent cosmid transfectedcells presenting P815A antigen were isolated. The DNA of thesetransfectants was isolated and packaged directly with lambda phageextracts, following DePlaen et al., Proc. Natl. Acad. Sci. USA 85:2274-2278 (1988). The resulting product was titrated on E. coli ED 8767with ampicillin selection, as in Example 5. Similarly, amplification ofthe cosmids and transfection followed Example 5, again using PO.HTR.

High frequencies of transfection were observed, as described in Table 1,which follows:

TABLE 1 Transfer of the expression of antigen P815A by cosmids obtainedby direct packaging Transfectant No. of transfectants obtained with No.of cosmids obtained expressing P815A/ the cosmid by direct packaging ofno. of HmB^(r) library 0.5 μg of DNA transfectants TC3.1 32 87/192 TC3.232000 49/384 TC3.3 44 25/72

The cosmids were analyzed with restriction enzymes and it was found thatdirectly packaged transfectant P1A.TC3.1 contained 32 cosmids, 7 ofwhich were different. Each of these 7 cosmids was transfected intoPO.HTR, in the manner described supra, and again, following theprotocols described above, transfectants were studied for presentationof P815A. Four of the cosmid transfectants showed P815A presentationand, as with all experiments described herein, P815B was co-expressed.

Of the four cosmids showing presentation of the two antigens, cosmidCIA.3.1 was only 16.7 kilobases long, and was selected for furtheranalysis as described infra.

The cosmid C1A.3.1 was subjected to restriction endonuclease analysis,yielding the map shown in FIG. 3.

All EcoRI fragments were transfected, again using the above describedprotocols, and only the 7.4 kilobase fragment produced a transfectantthat anti-A CTLs could lyse. Similar experiments were carried out on thePstI fragments, and only a 4.1 kb fragment fully contained within the7.4 kb. EcoRI fragment produced lysable transfectants.

This fragment (i.e., the 4.1 kb PstI fragment), was digested with SmaI,giving a 2.3 kb fragment which also yielded host cells presentingantigens A and B after transfection. Finally, a fragment 900 bases long,secured with SmaI/XbaI, also transferred expression of the precursors ofthese two antigens, i.e., the transfected host cell presented bothantigen A and antigen B.

EXAMPLE 7

The 900 base fragment described above was used as a probe to detect theexpression of the P815A gene in parent cell line P1.HTR. To accomplishthis, total cellular RNA was first isolated using theguanidine-isothiocyanate procedure of Davis et al., Basic Methods InMolecular Biology (Elseview Science Publishing Co, New York) (1986). Thesame reference was the source of the method used to isolate and purifypolyA⁺ mRNA using oligodT cellulose column chromatography.

Samples were then subjected to Northern Blot analysis. RNA samples werefractionated on 1% agarose gels containing 0.66 M formaldehyde. The gelswere treated with 10×SSC (SSC: 0.15 M NaCl; 0.015 M sodium citrate, pH7.0) for 30 minutes before overnight blotting on nitrocellulosemembranes. These were baked for two hours at 80° C., after which themembranes were prehybridized for 15 minutes at 60° C. in a solutioncontaining 10% dextran sulfate, 1% SDS and 1M NaCl. Hybridization wasthen carried out using denatured probe (the 900 base fragment), togetherwith 100 ug/ml salmon sperm DNA.

When this protocol was carried out using P1.HTR poly A⁺ RNA, a band of1.2 kb and two fainter bands were identified, as shown in FIG. 4, lane 1(6 ug of the RNA).

The same probe was used to screen a cDNA library, prepared from poly-A⁺RNA from the cell line. This yielded a clone with a 1 kb insert,suggesting a missing 5′ end. The Northern blots for the cDNA are notshown.

Hybridization experiments in each case were carried out overnight at 60°C. The blots were washed twice at room temperature with 2×SSC and twiceat 60° C. with 2×SSC supplemented with 1% SDS.

The foregoing experiments delineated the DNA expressing the P815Aantigen precursor sufficiently to allow sequencing, using the well knownSanger dideoxy chain termination method. This was carried out on clonesgenerated using a variety of restriction endonucleases and by specificpriming with synthetic oligonucleotide primers. The results for exons ofthe gene are set forth in sequence id no: 4.

EXAMPLE 8

The Northern analysis described supra suggested that the 5′ end of thecDNA was missing. To obtain this sequence, cDNA was prepared from P1.HTRRNA using a primer corresponding to positions 320-303. The sequence wasthen amplified using the polymerase chain reaction using a 3′ primercorresponding to positions 286-266 and a 5′ primer described by Frohmanet al., Proc. Natl. Acad. Sci. USA 85: 8998-9002 (1988). A band of theexpected size (270 bases) was found, which hybridized to the 900 bpSmaI/XbaI fragment described supra on a Southern blot. Following cloninginto m13tg 130 λ tg 131, the small, 270 bp fragment was sequenced. Thesequence is shown in sequence id no: 1.

EXAMPLE 9

Following the procurement of the sequences described in Examples 7 and 8and depicted in seq id no: 4, a 5.7 kb region of cosmid CIA.3.1 wassequenced. This fragment was known to contain the 900 base fragmentwhich expressed P815A in transfectants. This experiment permitteddelineation of introns and exons, since the cosmid is genomic in origin.

The delineated structure of the gene is shown in FIG. 5. Together withseq id no: 4, these data show that the gene for the antigen precursor,referred to as “P1A” hereafter, is approximately 5 kilobases long andcontains 3 exons. An ORF for a protein of 224 amino acids starts in exon1, ending in exon 2. The 900 base pair fragment which transfersexpression of precursors for antigens A and B only contains exon 1. Thepromoter region contains a CAAT box, as indicated in seq. id no: 1, andan enhancer sequence. This latter feature has been observed in promotersof most MHC class I genes, as observed by Geraghty et al., J. Exp. Med171: 1-18 (1990); Kimura et al., Cell 44: 261-272 (1986).

A computer homology search was carried out, using program FASTA withK-triple parameters of 3 and 6, as suggested by Lipman et al., Science227: 1435-1441 (1985), and using Genbank database release 65 (October1990). No homology was found except for a stretch of 95 basescorresponding to part of an acid region coded by exon 1 (positions524-618), which is similar to sequences coding for acidic regions inmouse nucleolar protein N038/B23, as described by Bourbon et al., Mol.Biol. 200: 627-638 (1988), and Schmidt-Zachmann et al., Chromosoma 96:417-426 (1988). Fifty six of 95 bases were identical. In order to testwhether these homologies were the reason for cross hybridizing,experiments were carried out using a mouse spleen cDNA library screenedwith the 900 base fragment. cDNA clones corresponding closely to thesizes of the cross hybridizing bands were obtained. These were partiallysequenced, and the 2.6 kb cDNA was found to correspond exactly toreported cDNA sequence of mouse nucleolin, while the 1.5 kb cDNAcorresponded to mouse nucleolar protein NO38/B23.

Analysis of the nucleotide sequence of the gene, referred to as “P1A”hereafter, suggests that its coded product has a molecular mass of 25kd. Analysis of the sequence id no: 4 shows a potential nucleartargeting signal at residues 5-9 (Dingwall et al., Ann. Rev. Cell Biol.2: 367-390 (1986)), as well as a large acidic domain at positions83-118. As indicated supra, this contains the region of homology betweenP1A and the two nucleolar proteins. A putative phosphorylation site canbe found at position 125 (serine). Also, a second acidic domain is foundclose to the C-terminus as an uninterrupted stretch of 14 glutamateresidues. A similar C-terminal structure has been found by Kessel et al.Proc. Natl. Acad. Sci. USA 84: 5306-5310 (1987), in a murine homeodomainprotein having nuclear localization.

In studies comparing the sequence of gene P1A to the sequences for P91A,35B and P198, no similarities were found; showing that P1A is indicativeof a different class of genes and antigens.

EXAMPLE 10

With the P1A probe and sequence in hand, investigations were carried outto determine whether the gene present in normal tissue was identical tothat expressed by the tumor. To do this, phage libraries were prepared,using lambda zapII 10 and genonic DNA of DBA2 murine kidney cells. P1Awas used as a probe. Hybridization conditions were as described supra,and a hybridizing clone was found. The clone contained exons one and twoof the P1A gene, and corresponded to positions −0.7 to 3.8 of FIG. 5.Following localization of this sequence, PCR amplification was carriedout to obtain the sequence corresponding to 3.8 to 4.5 of FIG. 5.

Sequence analysis was carried out, and no differences were found betweenthe gene from normal kidneys and the P1A gene as obtained from the P815tumor cells.

In further experiments, the gene as found in DBA/2 kidney cells wastransfected into PO.HTR, as described supra. These experiments,presented pictorially in FIG. 7, showed that antigens A and B wereexpressed as efficiently by the kidney gene isolated from normal kidneycells as with the P1A gene isolated from normal kidney cells.

These experiments lead to the conclusion that the gene coding for thetumor rejection antigen precursor is a gene that does not result from amutation; rather, it would appear that the gene is the same as onepresent in normal cells, but is not expressed therein. The ramificationsof this finding are important, and are discussed infra.

In studies not elaborated upon herein, it was found that variants of thegene were available. Some cells were “P1A⁻B⁺”, rather than the normal“P1A”. The only difference between these is a point mutation in exon 1,with the 18th triplet coding for Ala in the variant instead of Val.

EXAMPLE 11

Additional experiments were carried out with other cell types. Followingthe protocols described for Northern blot hybridizations supra, RNA ofnormal liver and spleen cells was tested to determine if a transcript ofthe P1A gene could be found. The Northern blot data are presented inFIG. 4 and, as can be seen, there is no evidence of expression.

The murine P815 cell line from which P1A was isolated is a mastocytoma.Therefore, mast cell lines were studied to determine if they expressedthe gene. Mast cell line MC/9, described by Nabel et al., Cell 23: 19-28(1981), and short term cultures of bone marrow derived mast cells weretested in the manner described supra (Northern blotting), but notranscript was found. In contrast when a Balb/C derived IL-3 dependentcell line L138.8A (Hültner et al., J. Immunol. 142: 3440-3446 (1989))was tested, a strong signal was found. The mast cell work is shown inFIG. 4.

It is known that both BALB/C and DBA/2 mice share H-2^(d) haplotype, andthus it was possible to test sensitivity to lysis using the CTLsdescribed supra. FIG. 8 shows these results, which essentially provethat anti-A and anti-B CTLs lysed the cells strongly, whereas anti-C andanti-D lines did not.

Further tests were carried out on other murine tumor cell lines, i.e.,teratocarcinoma cell line PCC4 (Boon et al., Proc. Natl. Acad. Sci. USA74: 272-275 (1977), and leukemias LEC and WEH1-3B. Expression could notbe detected in any of these samples.

EXAMPLE 12

The actual presentation of the P1A antigen by MHC molecules was ofinterest. To test this, cosmid C1A.3.1 was transfected into fibroblastcell line DAP, which shows phenotype H-₂ ^(k). The cell lines weretransfected with genes expressing one of the K^(d), D_(d), and L^(d)antigen. Following transfection with both the cosmid and the MHC gene,lysis with CTLs was studied, again as described supra. These studies,summarized in Table 2, show that L^(d) is required for presentation ofthe P1A antigens A and B.

TABLE 2 H-2-restriction of antigens P815A ind P815B No. of clones lysedby the CTL/no. of HmB^(r) clones^(≠) Recipient cell* CTL anti-A CTLanti-B DAP (H-2^(k)) 0/208 0/194 DAP + K^(d) 0/165 0/162 DAP + D^(d)0/157 0/129 DAP + L^(d) 25/33  15/20  *Cosmid C1A.3.I containing theentire P1A gene was transfected in DAP cells previously transfected withH-2^(d) class I genes as indicated. ^(≠)Independent drug-resistantcolonies were tested for lysis by anti-A or anti-B CTL in a visualassay.

The observation that one may associate presentation of a tumor rejectionantigen with a particular MHC molecule was confirmed in experiments withhuman cells and HLA molecules, as elaborated upon infra.

EXAMPLE 13

Using the sequence of the P1A gene as well as the amino acid sequencederivable therefrom, antigenic peptides which were A⁺ B⁺ (i.e.,characteristic of cells which express both the A and B antigens), andthose which are A⁻B⁺ were identified. The peptide is presented in FIG.10. This peptide when administered to samples of PO.HTR cells in thepresence of CTL cell lines specific to cells presenting it, led to lysisof the PO.HTR cells, lending support to the view that peptides based onthe product expressed by the gene can be used as vaccines.

EXAMPLE 14

The human melanoma cell line referred to hereafter as MZ2-MEL is not aclonal cell line. It expresses four stable antigens recognized byautologous CTLS, known as antigens “D, E, F, and A”. In addition, twoother antigens “B” and “C” are expressed by some sublines of the tumor.CTL clones specific for these six antigens are described by Van denEynde et al., Int. J. Canc. 44: 634-640 (1989). Among the recognizedsubclones of MZ2-MEL are MEL.43, MEL3.0 and MEL3.1. (Van den Eynde etal., supra). Cell line MEL3.1 expresses antigen E, as determined by CTLstudies as described for P815 variants, supra, so it was chosen as asource for the nucleic acid sequence expressing the antigen precursor.

In isolating the pertinent nucleic acid sequence for a tumor rejectionantigen precursor, the techniques developed supra, showed that arecipient cell is needed which fulfills two criteria: (i) the recipientcell must not express the TRAP of interest under normal conditions, and(ii) it must express the relevant class I HLA molecule. Also, therecipient cell must have a high transfection frequency, i.e., it must bea “good” recipient.

In order to secure such a cell line, the clonal subline ME3.1 wassubjected to repeated selection with anti-E CTL 82/30 as described byVan den Eynde, supra. The repeated cycles of selection led to isolationof subclone MZ2-MEL-2.2 isc E³¹ . This subclone is also HPRT⁻, (i.e.,sensitive to HAT medium: 10⁻⁴ M hypoxanthine, 3.8×10⁻⁷ aminopterine,1.6×10⁻⁵ M 2-deoxythymidine). The subclone is referred to as “MEL-2.2”for simplicity hereafter.

EXAMPLE 15

The genomic DNA of MEL3.0 was prepared following Wölfel et al.,Immunogenetics 26: 178-187 (1987), the disclosure of which isincorporated by reference. The plasmid pSVtkneoβ, as described byNicolas et al., Cold Spring Harb., Conf. Cell Prolif. 10: 469-485 (1983)confers geneticin resistance, so it can be used as a marker forcotransfection, as it was in this experiment.

Following a procedure similar but not identical to that of Corsao etal., Somatic Cell Molec. Genet 7: 603-616 (1981), total genomic DNA andthe plasmid were cotransfected. The genomic DNA (60 μg) and plasmid DNA(6 μg) were mixed in 940 μl of 1 mM Tris.HCl (pH 7.5), 0.1 mM EDTA,after which 310 μl of 1M CaCl₂ was added. This solution was slowlyadded, under constant agitation, to 1.25 ml of 2×HBS (50 mM HEPES, 280mM NaCl 1.5 mM Na₂HPO₄, adjusted to pH 7.1 with NaOH). The calciumphosphate DNA precipitates were allowed to form for 30-45 minutes atroom temperature, after which they were applied to 80 cm² tissue cultureflasks which had been seeded 24 hours previously with 3×10⁶ MEL2.2cells, in 22.5 ml of melanoma culture medium (Dulbecco's ModifiedEagle's Medium) supplemented with 10% fetal calf serum. After 24 hours,the medium was replaced. Forty eight hours after transfection, the cellswere harvested and seeded at 4×10⁶ cells per 80 cm² flask in melanomaculture medium supplemented with 2 mg/ml of geneticin. The geneticinserves as a selection marker.

EXAMPLE 16

Thirteen days after transfection, geneticin-resistant colonies werecounted, harvested, and cultured in nonselective medium for 2 or 3 days.Transfected cells were then plated in 96-well microplates at 200cells/well in 200 ul of culture medium with 20% fetal calf serum (FCS)in order to obtain approximately 30 growing colonies per well. Thenumber of microcultures was aimed at achieving redundancy, i.e., suchthat every independent transfectant should be represented at least fourtimes.

After 10 days, wells contained approximately 6×10⁴ cells. These cellswere detached, and ⅓ of each microculture was transferred to a duplicateplate. After 6 hours, i.e., after readherence, medium was removed and1500 anti-E CTL (CTL 82/30), were added to each well in 100 μl of CTLculture medium with 35 U/ml of IL-2. One day later, the supernatant (50μl) was harvested and examined for TNF concentration, for reasons setforth in the following example.

EXAMPLE 17

The size of the mammalian genome is 6×10⁶ kb. As the average amount ofDNA integrated in each drug-resistant transfectant was expected to beabout 200 kb, a minimum of 30,000 transfectants would need to beexamined to ascertain whether antigen E had been transfected. Prior workwith murine cells had shown that when a CTL stimulation assay was used,groups containing only 3% of cells expressing the antigen of interestedcould be identified. This should reduce the number of assays by a factorof 30. While an anti-E CTL assay, as described supra, in mixed E⁺/E⁻cells was helpful, it was not sufficient in that consistent resultscould not be obtained.

As a result, an alternative test was devised. Stimulation of CTLs wasstudied by release of tumor necrosis factor (“TNF”) using well knownmethodologies which need not be repeated here. As described in Example15, 1500 CTL 82/30 cells had been added per well of transfectants. TheseCTLs were collected 6 days after stimulation. As indicated supra, after⅓ of the cells in each well had been removed and the remaining ⅔ (4×10⁴)had readhered, the CTLs and IL-2 were added thereto. The 50 μl ofsupernatant was removed 24 hours later and transferred to a microplatecontaining 3×10⁴ W13 (WEHI-164 clone 13; Espevik et al., J. Immunol.Meth. 95: 99-105 (1986)) cells in 50 μl of W13 culture medium(RPMI-1640, supplemented with L-arginine (116 mg/l), L-asparagine (36mg/l), L-glutamine (216 mg/l), and 10% FCS supplemented with 2 μg ofactinomycin D at 37% in an 8% Co₂ atmosphere. The cell line W13 is amouse fibrosarcoma cell line sensitive to TNF. Dilutions of recombinantTNF-β in RPMI 1640 were added to target cell controls.

The W13 cultures were evaluated after 20 hours of incubation, and deadcell percentage was measured using an adaptation of the colorimetricassay of Hansen et al., J. Immunol. Meth. 119: 203-210 (1989). Thisinvolved adding 50 ml of (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide at 2.5 mg/ml in PBS, followed by two hours ofincubation at 37° C. Dark blue formazan crystals were dissolved byadding 100 μl of lysis solution (1 volume N,N dimethyl formamide mixedat 37° C. with two volumes of water containing 30% (w/v) sodium dodecylsulphate, at pH 4.7 from 1.6% acetic acid and 2.5% 1N HCl). Plates wereincubated at 37° C. overnight, and ODs were taken at 570 nm using 650 nmas control. Dead cell percentage was determined via the formula:$100 \times \left\lbrack {1 - \frac{100 - \left( {{OD}_{570}\quad {sample}{\quad \quad}{well}} \right)}{{{OD}_{570}\quad {well}} + {medium}}} \right\rbrack$

following Espevik et al., J. Immunol. Meth. 95: 99-105 (1986). Theresults showed that even when the ratio of E⁺/E⁻ cells was as low as{fraction (1/45)}, significant production of TNF was observed, thusshowing active CTLs. This led to the decision to test the drug resistanttransfectants in groups of 30.

EXAMPLE 18

Cells were tested for TNF production as discussed in Example 17, supra.A total of 100 groups of E⁻ cells (4×10⁶ cells/group) were testedfollowing transfection, and 7×10⁴ independent geneticin resistanttransfectants were obtained, for an average of 700 per group. Only onegroup of transfected cells led to a microculture which caused anti-Eantigen CTL clone 82/30 to produce TNF. Of 300 clones tested, 8 werepositive. These clones were then tested for lysis by anti-E CTL, usingthe standard ⁵¹Cr release assay, and were found to be lysed asefficiently as the original E⁺ cell line. The transfectant E.T1,discussed herein, had the same lysis pattern as did MEL2.2 for CTLsagainst antigens B,C,D and F.

The fact that only one transfectant presented the antigen out of 70,000geneticin resistance transfectants may at first seem very low, but it isnot. The work described supra for P815 showed an average frequency of{fraction (1/13,000)}. Human DNA recipient MEL2.2 appears to integrate 5times less DNA than P1.HTR.

EXAMPLE 19

Once transfectant E.T1 was found, analysis had to address severalquestions including whether an E⁺ contaminant of the cell population wasthe cause. The analysis of antigen presentation, described supra, showsthat E.T1 is B⁻ and C⁻, just like the recipient cell MEL2.2. It was alsofound to be HPRT⁻, using standard selection procedures. All E⁺ cellsused in the work described herein, however, were HPRT⁺.

It was also possible that an E⁺ revertant of MEL2.2 was the source forE.T1. To test this, the observation by Perucho et al., Cell 22: 309-317(1980), that cotransfected sequences usually integrate together at asingle location of recipient genome was employed. If antigen E in atransfectant results from cotransfection with pSVtkneoβ, then sequencesshould be linked and deletion of the antigen might also delete theneighboring pSVtkneoβ sequences. Wölfel et al., supra, has shown this tobe true. If a normally E⁻ cell is transfected with pSVtkneoβ, thensequences should be linked and deletion of the antigen might also deletethe neighboring pSVtkneoβ sequences. If a normally E⁺ cell transfectedwith pSVtkneoβ is E.T1, however, “co-deletion” should not take place. Totest this, the transfectant E.T1 was subjected to immunoselection with82/30, as described supra. Two antigen loss variants were obtained,which resisted lysis by this CTL. Neither of these had lost geneticinresistance; however, Southern blot analysis showed loss of severalneo^(r) sequences in the variants, showing close linkage between the Egene and neo^(r) gene in E.T1, leading to the conclusion that E.T1 was atransfectant.

EXAMPLE 20

The E⁺ subclone MZ2-MEL 4B was used as a source of DNA for preparationof a cosmid library. This library of nearly 700,000 cosmids wastransfected into MZ2-MEL 2.2 cells, following the cosmid transfectionprotocols described supra.

By packaging the DNA of cosmid transfectants directly into lambda phasecomponents, it is sometimes possible to retrieve cosmids that containthe sequences of interest. This procedure was unsuccessful here, so werescued the transfected sequence by ligating DNA of the transfectant toappropriate restriction fragments of cosmid vector pTL6. This was triedwith two transfectants and was successful with one of them. One cosmid,referred to as B3, was recovered from this experiment, and subjected torestriction endonuclease digestion via XmaI, or by BamHI digestion of alarge, 12 kb XmaI transfected fragment. The fragments were cloned intovector pTZ 18R, and then transfected into MEL2.2. Again, TNF productionwas the measure by which successful transfection was determined. Theexperiments led to the determination of a gene sequence capable oftransfecting antigen E on the 12 kb XmaI fragment, and then on the 2.4kb fragment of BamHI digestion of the 12 kb segment.

The 2.4 kb fragment hybridizes with a 2.4 kb fragment from MZ2-MEL andwith a T cell clone of patient MZ-2, as determined by Southern Blots(BamHI/SmaI digested DNA). The band is absent from E⁻ antigen lossvariants of MZ2-MEL, as seen in FIG. 12.

The sequence for the E antigen precursor gene has been determined and ispresented herein and as SEQ ID NO:7.

         |   10     |   20     |   30     |   40     |   50     |   60   1 GGATCCAGGC CCTGCCAGGA AAAATATAAG GGCCCTGCGT GAGAACAGAG GGGGTCATCC61   61 ACTGCATGAG AGTGGGGATG TCACAGAGTC CAGCCCACCC TCCTGGTAGCACTGAGAAGC 121  121 CAGGGCTGTG CTTGCGGTCT GCACCCTGAG GGCCCGTGGATTCCTCTTCC TGGAGCTCCA 181  181 GGAACCAGGC AGTGAGGCCT TGGTCTGAGACAGTATCCTC AGGTCACAGA GCAGAGGATG 241  241 CACAGGGTGT GCCAGCAGTGAATGTTTGCC CTGAATGCAC ACCAAGGGCC CCACCTGCCA 301  301 CAGGACACATAGGACTCCAC AGAGTCTGGC CTCACCTCCC TACTGTCAGT CCTGTAGAAT 361  361CGACCTCTGC TGGCCGGCTG TACCCTGAGT ACCCTCTCAC TTCCTCCTTC AGGTTTTCAG 421 421 GGGACAGGCC AACCCAGAGG ACAGGATTCC CTGGAGGCCA CAGAGGAGCA CCAAGGAGAA481  481 GATCTGTAAG TAGGCCTTTG TTAGAGTCTC CAAGGTTCAG TTCTCAGCTGAGGCCTCTCA 541  541 CACACTCCCT CTCTCCCCAG GCCTGTGGGT CTTCATTGCCCAGCTCCTGC CCACACTCCT 601  601 GCCTGCTGCC CTGACGAGAG TCATCATGTCTCTTGAGCAG AGGAGTCTGC ACTGCAAGCC 661  661 TGAGGAAGCC CTTGAGGCCCAACAAGAGGC CCTGGGCTGG TGTGTGTGCA GGCTGCCACC 721  721 TCCTCCTCCTCTCCTCTGGT CCTGGGCACC CTGGAGGAGG TGCCCACTGC TGGGTCAACA 781  781GATCCTCCCC AGAGTCCTCA GGGAGCCTCC GCCTTTCCCA CTACCATCAA CTTCACTCGA 841 841 CAGAGGCAAC CCAGTGAGGG TTCCAGCAGC CGTGAAGAGG AGGGGCCAAG CACCTCTTGT901  901 ATCCTGGAGT CCTTGTTCCG AGCAGTAATC ACTAAGAAGG TGGCTGATTTGGTTGGTTTT 961  961 CTGCTCCTCA AATATCGAGC CAGGGAGCCA GTCACAAAGGCAGAAATGCT GGAGAGTGTC 1021 1021 ATCAAAAATT ACAAGCACTG TTTTCCTGAGATCTTCGGCA AAGCCTCTGA GTCCTTGCAG 1081 1081 CTGGTCTTTG GCATTGACGTGAAGGAAGCA GACCCCACCG GCCACTCCTA TGTCCTTGTC 1141 1141 ACCTGCCTAGGTCTCTCCTA TGATGGCCTG CTGGGTGATA ATCAGATCAT GCCCAAGACA 1201 1201GGCTTCCTGA TAATTGTCCT GGTCATGATT GCAATGGAGG GCGGCCATGC TCCTGAGGAG 12611261 GAAATCTGGG AGGAGCTGAG TGTGATGGAG GTGTATGATG GGAGGGAGCA CAGTGCCTAT1321 1321 GGGGAGCCCA GGAAGCTGCT CACCCAAGAT TTGGTGCAGG AAAAGTACCTGGAGTACGGC 1381 1381 AGGTGCCGGA CAGTGATCCC GCACGCTATG AGTTCCTGTGGGGTCCAAGG GCCCTCGCTG 1441 1441 AAACCAGCTA TGTGAAAGTC CTTGAGTATGTGATCAAGGT CAGTGCAAGA GTTCGCTTTT 1501 1501 TCTTCCCATC CCTGCGTGAAGCAGCTTTGA GAGAGGAGGA AGAGGGAGTC TGAGCATGAG 1561 1561 TTGCAGCCAAGGCCAGTGGG AGGGGGACTG GGCCAGTGCA CCTTCCAGGG CCGCGTCCAG 1621 1621CAGCTTCCCC TGCCTCGTGT GACATGAGGC CCATTCTTCA CTCTGAAGAG AGCGGTCAGT 16811681 GTTCTCAGTA GTAGGTTTCT GTTCTATTGG GTGACTTGGA GATTTATCTT TGTTCTCTTT1741 1741 TGGAATTGTT CAAATGTTTT TTTTTAAGGG ATGGTTGAAT GAACTTCAGCATCCAAGTTT 1801 1801 ATGAATGACA GCAGTCACAC AGTTCTGTGT ATATAGTTTAAGGGTAAGAG TCTTGTGTTT 1861 1861 TATTCAGATT GGGAAATCCA TTCTATTTTGTGAATTGGGA TAATAACAGC AGTGGAATAA 1921 1921 GTACTTAGAA ATGTGAAAAATGAGCAGTAA AATAGATGAG ATAAAGAACT AAAGAAATTA 1981 1981 AGAGATAGTCAATTCTTGCC TTATACCTCA GTCTATTCTG TAAAATTTTT AAAGATATAT 2041 2041GCATACCTGG ATTTCCTTGG CTTCTTTGAG AATGTAAGAG AAATTAAATC TGAATAAAGA 21012101 ATTCTTCCTG TTCACTGGCT CTTTTCTTCT CCATGCACTG AGCATCTGCT TTTTGGAAGG2161 2161 CCCTGGGTTA GTAGTGGAGA TGCTAAGGTA AGCCAGACTC ATACCCACCCATAGGGTCGT 2221 2221 AGAGTCTAGG AGCTGCAGTC ACGTAATCGA GGTGGCAAGATGTCCTCTAA AGATGTAGGG 2281 2281 AAAAGTGAGA GAGGGGTGAG GGTGTGGGGCTCCGGGTGAG AGTGGTGGAG TGTCAATGCC 2341 2341 CTGAGCTGGG GCATTTTGGGCTTTGGGAAA CTGCAGTTCC TTCTGGGGGA GCTGATTGTA 2401 2401 ATGATCTTGGGTGGATCC                                               2418         |   10     |   20     |   30     |   40     |   50     |   60

EXAMPLE 21

After the 2.4 kb genomic segment had been identified, studies werecarried out to determine if an “E⁺” subline expressed any homologousDNA. Cell line MZ2-MEL 3.0 was used as a source, and a cDNA library wasprepared from its mRNA, using art known techniques. The 2.4 kb segmentwas used as a probe, and MRNA of about 1.8 kb was identified ashomologous, using Northern blot analysis. When cDNA was screened, cloneswere obtained showing almost complete identity to parts of the 2.4 kbfragment. Two exons were thus identified. An additional exon was locatedupstream of these, via sequencing segments of cosmid B3 located in frontof the 2.4 kb BamHI fragment. The gene extends over about 4.5 kb, asshown in FIG. 8. The starting point of the transcribed region wasconfirmed using PCR for the 5′ end of the cDNA. The three exons comprise65, 73, and 1551 base pairs. An ATG is located at position 66 of exon 3,followed by an 828 base pair reading frame.

EXAMPLE 22

To determine if smaller segments of the 2.4 kb fragment could transferthe expression of antigen E, smaller pieces corresponding to the largergene were prepared, using art recognized techniques, and transferredinto E⁻ cells. FIG. 8 shows the boundaries of the three segments.

Transfer of antigen expression in this manner indicates that the genecodes for the antigen precursor, rather than coding for a protein whichactivates the antigen.

EXAMPLE 23

The probing of cDNA described supra revealed, surprisingly, twodifferent but closely related cDNAs. These cDNAs, when tested, did nottransfer expression of antigen E, but they do show substantial homologyto the first cDNA segment. The three segments, appear to indicate anewly recognized family of genes, referred to as “MAGE” for “melanomaantigen”. In FIG. 9, “mage-1” directs expression of the antigen from MZ2cells. Portions of the third exon of each gene are presented in FIG. 9.The second and third sequences are more closely related to each otherthan the first (18.1 and 18.9% difference compared to the first; 12%with each other). Out of 9 cDNA clones obtained, three of each type wereobtained, suggesting equal expression. “MAGE” as used hereafter refersto a family of molecules, and the nucleic acids coding for them. Thesenucleic acids share a certain degree of homology and are expressed intumor cells including several types of human tumor cells as well as inhuman tumors. The family is referred to as “MAGE” because the firstmembers were identified in human melanoma cells. As the experimentswhich follow indicate, however, the members of the MAGE family are notat all restricted to melanoma tumors; rather, MAGE refers to a family oftumor rejection antigen precursors and the nucleic acid sequences codingtherefore. The antigens resulting therefrom are referred to herein as“MAGE TRAs” or “melanoma antigen tumor rejection antigens”

EXAMPLE 24

Experiments with mouse tumors have demonstrated that new antigensrecognized by T cells can result from point mutations that modify activegenes in a region that codes for the new antigenic peptide. New antigenscan also arise from the activation of genes that are not expressed inmost normal cells. To clarify this issue for antigen MZ2-E, the mage-1gene present in the melanoma cells was compared to that present innormal cells of patient MZ2. Amplification by polymerase chain reaction(PCR) of DNA of phytohemagglutinin-activated blood lymphocytes usingprimers surrounding a 1300 bp stretch covering the first half of the 2.4kb fragment was carried out. As expected, a PCR product was obtainedwhereas none was obtained with the DNA of the E⁻ variant. The sequenceof this PCR product proved identical to the corresponding sequence ofthe gene carried by the E⁺ melanoma cells. Moreover, it was found thatantigen MZ2-E was expressed by cells transfected with the cloned PCRproduct. This result suggests that the activation of a gene normallysilent is responsible for the appearance of tumor rejection antigenMZ2-E.

EXAMPLE 25

In order to evaluate the expression of gene mage-1 by various normal andtumor cells, Northern blots were hybridized with a probe covering mostof the third exon. In contrast with the result observed with human tumorcell line MZ2-MEL 3.0, no band was observed with RNA isolated from a CTLclone of patient MZ2 and phytohemagglutinin-activated blood lymphocytesof the same patient. Also negative were several normal tissues of otherindividuals (FIG. 10 and FIG. 11). Fourteen melanoma cell lines of otherpatients were tested. Eleven were positive with bands of varyingintensities. In addition to these culture cell lines, four samples ofmelanoma tumor tissue were analyzed. Two samples, including a metastasisof patient MZ2 proved positive, excluding the possibility thatexpression of the gene represented a tissue culture artefact. A fewtumors of other histological types, including lung tumors were tested.Most of these tumors were positive (FIGS. 10 and 11). These resultsindicated that the MAGE gene family is expressed by many melanomas andalso by other tumors. However, they provided no clear indication as towhich of genes mage-1, 2 or 3 were expressed by these cells, because theDNA probes corresponding to the three genes cross-hybridized to aconsiderable extent. To render this analysis more specific, PCRamplification and hybridization with highly specific oligo-nucleotideprobes were used. cDNAs were obtained and amplified by PCR usingoligonucleotide primers corresponding to sequences of exon 3 that wereidentical for the three MAGE genes discussed herein. The PCR productswere then tested for their ability to hybridize to three otheroligonucleotides that showed complete specificity for one of the threegenes (FIG. 9). Control experiments carried out by diluting RNA ofmelanoma MZ2-MEL 3.0 in RNA from negative cells indicated that under theconditions used herein the intensity of the signal decreasedproportionally to the dilution and that positive signals could still bedetected at a dilution of {fraction (1/300)}. The normal cells(lymphocytes) that were tested by PCR were confirmed to be negative forthe expression of the three MAGE genes, suggesting therefore a level ofexpression of less than {fraction (1/300)}^(th) that of the MZ2 melanomacell line (FIG. 11). For the panel of melanoma cell lines, the resultsclearly showed that some melanomas expressed MAGE genes mage 1, 2 and 3whereas other expressed only mage-2 and 3 (FIGS. 11 and 10). Some of theother tumors also expressed all three genes whereas others expressedonly mage-2 and 3 or only mage-3. It is impossible to exclude formallythat some positive PCR results do not reflect the expression of one ofthe three characterized MAGE genes but that of yet another closelyrelated gene that would share the sequence of the priming andhybridizing oligonucleotides. It can be concluded that the MAGE genefamily is expressed by a large array of different tumors and that thesegenes are silent in the normal cells tested to this point.

EXAMPLE 26

The availability of a sequence that transfects at high efficiency andefficiently expresses a TRAP made it possible to search for theassociated major histocompatibility complex (MHC) class I molecule. Theclass I specificities of patient MZ2 are HLA-A1, A29, B37, B44 and C6.Four other melanomas of patients that had A1 in common with MZ2 werecotransfected with the 2.4 kb fragment and pSVtkneoβ. Three of themyielded neo^(r) transfectants that stimulated TNF release by anti-E CTLclone 82/30, which is CD8+ (FIG. 10). No E− transfectant was obtainedwith four other melanomas, some of which shared A29, B44 or C6 with MZ2.This suggests that the presenting molecule for antigen MZ2-E is HLA-A1.In confirmation, it was found that, out of 6 melanoma cell lines derivedfrom tumors of HLA-A1 patients, two stimulated TNF release by anti-E CTLclone 82/30 of patient MZ2. One of these tumor cell lines, MI13443-MELalso showed high sensitivity to lysis by these anti-E CTL. These twomelanomas were those that expressed mage-1 gene (FIG. 13). Eightmelanomas of patients with HLA haplotypes that did not include A1 wereexamined for their sensitivity to lysis and for their ability tostimulate TNF release by the CTL. None was found to be positive. Theability of some human anti-tumor CTL to lyse allogeneic tumors sharingan appropriate HLA specificity with the original tumor has been reportedpreviously (Darrow, et al., J. Immunol. 142: 3329 (1989)). It is quitepossible that antigenic peptides encoded by genes mage 2 and 3 can alsobe presented to autologous CTL by HLA-A1 or other class I molecules,especially in view of the similar results found with murine tumors, aselaborated upon supra.

EXAMPLE 27

As indicated supra, melanoma MZ2 expressed antigens F, D and A′, inaddition to antigen E. Following the isolation of the nucleic acidsequence coding for antigen E, similar experiments were carried out toisolate the nucleic acid sequence coding for antigen F.

To do this, cultures of cell line MZ2-MEL2.2, an E⁻ cell line describedsupra, were treated with anti-F CTL clone 76/6, in the same mannerdescribed for treatment with anti-E CTL clones. This resulted in theisolation of an F antigen loss variant, which was then subjected toseveral rounds of selection. The resulting cell line, “MZ2-MEL2.2.5” wascompletely resistant to lysis by anti-F CTLs, yet proved to be lysed byanti-D CTLs.

Again, following the protocols set forth for isolation of antigen −Eprecursor DNA, the F⁻ variant was transfected with genomic DNA from F⁺cell line MZ2-MEL3.0. The experiments yielded 90,000 drug resistanttransfectants. These were tested for MZ2-F expression by using pools of30 cells in the TNF detection assay elaborated upon supra. One poolstimulated TNF release by anti-F CTLs, and was cloned. Five of 145clones were found to stimulate anti-F CTLs. Lysis assays, also followingprotocols described supra, confirmed (i) expression of the gene codingfor antigen F, and (ii) presentation of antigen F itself.

EXAMPLE 28

Following identification of F⁺ cell lines, the DNA therefrom was used totransfect cells. To do this, a cosmid library of F⁺ cell line MZ2-MEL.43was prepared, again using the protocols described supra. The library wasdivided into 14 groups of about 50,000 cosmids, and DNA from each groupwas transfected into MZ2-MEL2.2.5. Transfectants were then tested fortheir ability to stimulate TNF release from anti-F CTL clone 76/6. Of 14groups of cosmids, one produced two independent transfectants expressingantigen F; a yield of two positives out of 17,500 geniticin resistanttransfectants.

EXAMPLE 29

The existence of a gene family was suggested by the pattern observed onthe Southern blot (FIG. 12). To do this, the 2.4 kb BamHI fragment,which transferred the expression of antigen M22-E, was labelled with 32pand used as a probe on a Southern Blot of BamHI digested DNA of E +cloned subclone M22-MEL2.2. Hybridization conditions included 50 μl/cm²of 3.5×SSC, 1×Denhardt's solution; 25 mM sodium phosphate buffer (pH7.0), 0.5% SDS, 2mM EDTA, where the 2.4 kb probes had been labelled with[α³²p]dCTP (2-3000 Ci/mole), at 3×10⁶ cpm/ml. Hybridization was carriedout for 18 hours at 65° C. After this, the membranes were washed at 65°C. four times for one hour each in 2×SSC, 0.1% SDS, and finally for 30minutes in 0.1×SSC, 0.1% SDS. To identify hybridization, membranes wereautoradiographed using Kodak X-AR film and Kodak X-Omatic fineintensifying screens.

In the following examples, whenever “hybridization” is referred to, thestringency conditions used were similar to those described supra.“Stringent conditions” as used herein thus refers to the foregoingconditions; subject to routine, art recognized modification.

EXAMPLE 30

The cDNA coding for mage 4 was identified from a sample of the humansarcoma cell line LB23-SAR. This cell line was found to not express mage1, 2 or 3, but the mRNA of the cell line did hybridize to the 2.4 kbsequence for mage 1. To study this further, a cDNA library was preparedfrom total LB23-SAR mRNA, and was then hybridized to the 2.4 kbfragment. A cDNA sequence was identified as hybridizing to this probe,and is identified hereafter as mage 4.

EXAMPLE 31

Experiments were carried out using PHA-activated lymphocytes frompatient “MZ2”, the source of the “MZ” cells discussed supra. Anoligonucleotide probe which showed homology to mage 1 but not mage 2 or3 was hybridized with a cosmid library derived from the PHA activatedcells. The size of the hybridizing BamHI cosmid fragment, however, was4.5 kb, thus indicating that the material was not mage 1; however, onthe basis of homology to mage 1-4, the fragment can be referred to as“mage 5”. The sequence of MAGE 5 is presented in SEQ ID NO: 16.

EXAMPLE 32

Melanoma cell line LB-33 MEL was tested. Total mRNA from the cell linewas used to prepare cDNA, which was then amplified with oligos CH09:(ACTCAGCTCCTCCCAGATTT) (SEQ ID NO: 27), and CH010: (GAAGAGGAGGGGCCAAG)(SEQ ID NO: 28).

To do this, 1 μg of RNA was diluted to a total volume of 20 μl, using 2μl of 10×PCR buffer, 2 μl of each of 10 mM dNTP, 1.2 μl of 25 mM MgCl₂,1 μl of an 80 mM solution of CHO9, described supra, 20 units of RNAsin,and 200 units of M-MLV reverse transcriptase. This was followed byincubation for 40 minutes at 42° C. PCR amplification followed, using 8μl of 10×PCR buffer, 4.8 μl of 25 mM MgCl₂, 1 μl of CHO10, 2.5 units ofThermus acquaticus (“Taq”) polymerase, and water to a total volume of100 μl. Amplification was then carried out for 30 cycles (1 minute 94°C.; 2 minutes at 52° C., 3 minutes at 72° C.). Ten μl of each reactionwere then size fractionated on agarose gel, followed by nitrocelluloseblotting. The product was found to hybridize with oligonucleotide probeCHO18 (TCTTGTATCCTGGAGTCC). This probe identified mage 1 but not mage 2or 3. However, the product did not hybridize to probe SEQ 4(TTGCCAAGATCTCAGGAA). This probe also binds mage 1 but not 2 and 3. Thisindicated that the PCR product contained a sequence that differed frommage 1, 2 and 3. Sequencing of this fragment also indicated differenceswith respect to mage 4 and 5. These results indicate a sequencediffering from previously identified mage 1, 2, 3, 4 and 5, and is namedmage 6.

EXAMPLE 33

In additional experiments using cosmid libraries from PHA-activatedlymphocytes of MZ2, the 2.4 kb mage 1 fragment was used as a probe andisolated a complementary fragment. This clone, however, did not bind tooligonucleotides specific for mage 1, 2, 3 or 4. The sequence obtainedshows some homology to exon 3 of mage 1, and differs from mages 1-6. Itis referred to as mage 7 hereafter. Additional screenings yielded mage8-11.

EXAMPLE 34

The usefulness of the TRAPS, as well as TRAs derived therefrom, wasexemplified by the following.

Exon 3 of mage 1 was shown to transfer expression of antigen E. As aresult, it was decided to test whether synthetic peptides derived fromthis exon 3 could be used to confer sensitivity to anti-E CTL.

To do this, and using standard protocols, cells normally insensitive toanti-E/CTLs were incubated with the synthetic peptides derived from Exon3.1. Using the CTL lytic assays described supra on P815A, and a peptideconcentration of 3 mM, the peptide Glu-Ala-Asp-Pro-Thr-Gly-His-Ser-Tyr(SEQ ID NO: 26) was shown to be best. The assay showed lysis of 30%,indicating conferring of sensitivity to the anti-E CTL.

EXAMPLE 35

Nucleic acid sequences referred to as “smage” were isolated from murinecells. Using the protocols described supra, a cosmid library wasprepared from the DNA of normal DBA/2 kidney cells, using cosmid vectorC2RB. As a probe, the 2.4 kb BamHI fragment of MAGE-1 was used. The DNAwas blotted to nylon filters, and these were washed in 2×SSC at 65° C.to identify the smage material.

EXAMPLE 36

Further tissue samples were tested for the presence of MAGE genes, usingthe protocols discussed supra. Some of these results follow.

There was no expression of the MAGE genes in brain or kidney tumortissue. Colon tumor tissue showed expression of MAGE 1, 2, 3 and 4,although not all tumors tested showed expression of all MAGE genes. Thisis also true for pancreatic tumor (MAGE 1); non-small cell lung)(MAGE 1,2, 3 and 4), prostate (MAGE 1), sarcomas

(MAGE 1, 2, 3 and 4), breast (MAGE 1, 2,and 3), and larynx (MAGE 1 and4).

EXAMPLE 37

A cytolytic CTL clone “20/38” was obtained from peripheral bloodlymphocytes of melanoma patient MZ2. This clone is described by Van denEynde et al., Int. J. Cancer 44: 634-640 (1989), the disclosure of whichis incorporated by reference. The CTL clone has isolated following Herinet al., Int. J. Cancer 39: 390-396 (1987), which is incorporated byreference. The assay is described herein, however. Autologous melanomacells were grown in vitro, and then resuspended at 10⁷ cells/ml in DMEM,supplemented with 10% HEPES and 30% FCS, and incubated for 45 minutes at37° C. with 200 μCi/ml of Na(⁵¹Cr)O₄. Labelled cells were washed threetimes with DMEM, supplemented with 10 mM HEPES. These were thenresuspended in DMEM supplemented with 10 mM HEPES and 10% FCS, afterwhich 100 μl aliquots containing 10³ cells, were distributed into 96well microplates. Samples of the CTL clone were added in 100 ul of thesame medium, and assays were carried out in duplicate. Plates werecentrifuged for four minutes at 100 g, and incubated for four hours at37° C. in a 5.5% CO₂ atmosphere.

Plates were centrifuged again, and 100 ul aliquots of supernatant werecollected and counted. Percentage of ⁵¹Cr release was calculated asfollows:${{\% \quad}^{51}{Cr}\quad {release}} = {\frac{\left( {{ER} - {SR}} \right)}{\left( {{MR} - {SR}} \right)} \times 100}$

where ER is observed, experimental ⁵¹Cr release, SR is spontaneousrelease measured by incubating 10³ labeled cells in 200 ul of mediumalone, and MR is maximum release, obtained by adding 100 ul 0.3% TritonX-100 to target cells.

Those mononuclear blood samples which showed high CTL activity wereexpanded and cloned via limiting dilution, and were screened again,using the same methodology.

The same method was used to test target K562 cells. When EBV-B cellswere used, the only change was the replacement of DMEM medium by Hank'smedium, supplemented with 5% FCS.

These experiments led to isolation of CTL clone 20/38.

FIG. 1 presents the results of these assays. Specifically, it will beseen that the CTL clone lysed autologous melanoma cell line MZ2-MEL.3.0,but did not lyse EBV-B cell lines, fibroblasts, K562 or non-autologousmelanoma cell line SK-MEL-29.

EXAMPLE 38

Once the CTL clone was recognized as being specific for the autologouscell line, it was tested for antigenic specificity. To do this, antigenloss variants derived from patient MZ2 were tested in the same type ofchromium release assay described above. These target lines were MZ2-MEL3.0, which is D⁺, E⁺, F⁺, A⁺, MZ2-MEL.61, which is D⁻, MZ2-MEL 2.2,which is E⁻, and MZ2-MEL.4, which is F⁻. In addition to CTL clone 20/38,clones which are known to be anti-A (CTL 28/336), anti-F (CTL 76/6), andanti-E (CTL 22/13) were tested.

These results are set forth in FIG. 15. It will be seen that CTL clone20/38 lysed all the cell lines leading to chromium release except D⁻cell line MZ2-MEL.61, thus indicating that the CTL clone is anti-D. Thisresult was confirmed, in experiments not included herein, by experimentswhere TNF release by the CTL clone was observed only in the presence ofmelanoma lines presenting antigen D.

EXAMPLE 39

Once antigen D was identified as the target molecule, studies werecarried out to determine the HLA type which presented it. Theexperiments described in example A showed that antigen D was presentedby MZ2-MEL, and this cell line's HLA specificity is known (i.e., A1,A29, B37, B44, Cw6, C.cl.10). It was also known, however, that a variantof MZ2-MEL which had lost HLA molecules A29, B44 and C.cl.10 stillexpressed antigen D, so these could be eliminated from consideration.Studies were not carried out on lines expressing B37, as none could befound.

In all, 13 allogeneic lines were tested, which expressed either HLA-A1(10 of 13), or Cw6 (3 of 13). The cell lines were tested for theirability to stimulate release of TNF by CTL clone 20/38, using the methodof Traversari et al., Immunogenetics 35: 145-152 (1992), the disclosureof which is incorporated by reference. This assay measures TNF releasevia testing toxicity of supernatants on WEHI 164-13 cells.

In the assays, cell samples (3000, 10,000 or 30,000 cells) from theallogeneic lines were cultured in the presence of 1500 cells of the CTLclone, and 25 u/ml of IL-2. Twenty-four hours later, the supernatantfrom the culture was tested against the WEHI cells for toxicity. Theresults are presented in Table 1, which follows.

Eight cell lines were found to stimulate TNF release from the CTL clone20/38. All of these lines were HLA-A1. None of the Cw6 presenting linesdid so.

The cell lines were also assayed to determine MAGE expression. All eightof the lines which stimulated TNF release expressed MAGE-3, whereas thetwo HLA-A1 lines which were negative did not.

TABLE 3 TNF pg/ml Exp 1 Exp 2 Number +CTL +CTL Expression ExpressionMelanoma of Cells 20/38 20/38 of Mage-3 of HLA-A-1 MZ2-MEL.61.2 50000 14 +++ + MZ2-MEL-ET1 50000 >120 >120 +++ + 1666 66 >120 LY-1-MEL 300001 >120 1 >120 +++ + 10000 1 >120 1 >120 3000 <1 114 2 >120 MI-1022130000 <1 >120 +++ + 10000 <1 71 3000 <1 74 LY-2-MEL 30000 1 57 +++ +10000 1 86 3000 1 91 LY-4-MEL 30000 1 >120 +++ + 10000 1 >120 30001 >120 SK23-MEL 30000 1 112 +++ + 10000 1 116 3000 1 105 MI-665/2-MEL30000 1 3 2 4 − + 10000 1 2 2 5 3000 1 5, 2 1 5 LB34-MEL 30000 1 >120+++ + 10000 1 >120 3000 1 >120 LB45-MEL 30000 1 11 1 30 − + 10000 1 6 112 3000 1 2 <1 7 NA-6-MEL 30000 1 77 5 98 +++ + 10000 1 104 5 >120 30001 110 4 >120 MI-13443-KEL 30000 1 >120 +++ + 10000 1 >120 3000 1 >120LB5-MEL 30000 1 8 4 9 + − 10000 <1 5 4 11 3000 <1 5 1 5 SK64-MEL 30000 14 2 5 ? − 10000 1 2 1 5 3000 1 1 1 4 LB33-MEL 30000 1 3, 5 +++ − 10000 14 3000 1 3 LB73-MEL 50000 1500 CTL 20/38 and 25 μ/ml IL2 were mixed withthe indicated number of cells of the different allogeneic melanomas. 24hours later, the amount of TNF present in the supernatant was assayed bytesting its cytotoxicity for WEHI-164-13 cells.

EXAMPLE 40

In view of the results set forth in example C, experiments were carriedout to determine if antigen D was in fact a tumor rejection antigenderived from MAGE-3. To do this, recipient COS7 cells were transfectedwith 100 ng of the gene for HLA-A1 cloned into pcDNA I/Amp, and 100 ngof one of (a) cDNA for MAGE-1 cloned into pcDNA I/Amp, (b) cDNA forMAGE-2 cloned into pcDSRa, or (c) cDNA for MAGE-3 cloned into pcDSRa.The transfecting sequences were ligated into the plasmids in accordancewith manufacturer's instructions. Samples of COS-7 cells were seeded, at15,000 cells/well into tissue culture flat bottom microwells, inDulbeco's modified Eagles Medium (“DMEM”) supplemented with 10% fetalcalf serum. The cells were incubated overnight at 37° C., medium wasremoved and then replaced by 30 μl/well of DMEM medium containing 10% Nuserum, 400 μg/ml DEAE-dextran, 100 μM chloroquine, and the plasmidsdescribed above. Following four hours of incubation at 37° C., themedium was removed, and replaced by 50 μl of PBS containing 10% DMSO.This medium was removed after two minutes and replaced by 200 μl of DMEMsupplemented with 10% of FCS.

Following this change in medium, COS cells were incubated for 24 hoursat 37° C. Medium was then discarded, and 1500 cells of CTL clones 20/38were added, in 100 μl of Iscove medium containing 10% pooled humanserum, supplemented with 25 u/ml of IL-2. Supernatant was removed after24 hours, and TNF content was determined in an assay on WEHI cells, asdescribed by Traversari et al., Immunogenetics 35: 145-152 (1992), thedisclosure of which is incorporated by reference. These results areshown in FIG. 16.

It will be seen that the CTL clone was strongly stimulated by COS7 cellstransfected with HLA-A1 and MAGE-3, but not by the cells transfectedwith the other mage genes. This leads to the conclusion that antigen Dis a tumor rejection antigen derived from the tumor rejection antigenprecursor coded by gene MAGE-3, and that this TRA is presented by HLA-A1molecules.

The foregoing disclosure, including the examples, places many tools ofextreme value in the hands of the skilled artisan. To begin, theexamples identify and provide a methodology for isolating nucleic acidmolecules which code for tumor rejection antigen precursors as well asthe nucleic acid molecules complementary thereto. It is known that DNAexists in double stranded form, and that each of the two strands iscomplementary to the other. Nucleic acid hybridization technology hasdeveloped to the point where, given a strand of DNA, the skilled artisancan isolate its complement, or synthesize it.

“Nucleic acid molecule” as used herein refers to all species of DNA andRNA which possess the properties discussed supra. Genomic andcomplementary DNA, or “cDNA” both code for particular proteins, and asthe examples directed to isolation of MAGE coding sequences show, thisdisclosure teaches the artisan how to secure both of these.

Similarly, RNA molecules, such as mRNA can be secured. Again, withreference to the skilled artisan, once one has a coding sequence inhand, TRNA can be isolated or synthesized.

Complementary sequences which do not code for TRAP, such as “antisenseDNA” or mRNA are useful, e.g., in probing for the coding sequence aswell as in methodologies for blocking its expression.

It will also be clear that the examples show the manufacture ofbiologically pure cultures of cell lines which have been transfectedwith nucleic acid sequences which code for or express the TRAPmolecules. Such cultures can be used as a source for tumor rejectionantigens, e.g., or as therapeutics. This aspect of the invention isdiscussed infra.

Cells transfected with the TRAP coding sequences may also be transfectedwith other coding sequences. Examples of other coding sequences includecytokine genes, such as interleukins (e.g., IL-2 or IL-4), or majorhistocompatibility complex (MHC) or human leukocyte antigen (HLA)molecules. Cytokine gene transfection is of value because expression ofthese is expected to enhance the therapeutic efficacy of thebiologically pure culture of the cells in vivo. The art is well aware oftherapies where interleukin transfectants have been administered tosubjects for treating cancerous conditions. In a particularly preferredembodiment, cells are transfected with sequences coding for each of (i)a TRAP molecule, (ii) an HLA/MHC molecule, and (iii) a cytokine.

Transfection with an MHC/HLA coding sequence is desirable becausecertain of the TRAs may be preferentially or specifically presented onlyby particular MHC/HLA molecules. Thus, where a recipient cell alreadyexpresses the MHC/HLA molecule associated with presentation of a TRA,additional transfection may not be necessary although furthertransformation could be used to cause over-expression of the antigen. Onthe other hand, it may be desirable to transfect with a second sequencewhen the recipient cell does not normally express the relevant MHC/HLAmolecule. It is to be understood, of course, that transfection with oneadditional sequence does not preclude further transfection with othersequences.

The term “biologically pure” as used in connection with the cell linedescribed herein simply means that these are essentially free of othercells. Strictly speaking, a “cell line” by definition is “biologicallypure”, but the recitation will establish this fully.

Transfection of cells requires that an appropriate vector be used. Thus,the invention encompasses expression vectors where a coding sequence forthe TRAP of interest is operably linked to a promoter. The promoter maybe a strong promoter, such as those well known to the art, or adifferential promoter, i.e., one which is operative only in specificcell types. The expression vectors may also contain all or a part of aviral or bacterial genome, such as vaccinia virus or BCG. Such vectorsare especially useful in preparing vaccines.

The expression vectors may incorporate several coding sequences, as longas the TRAP sequence is contained therein. The cytokine and/or MHC/HLAgenes discussed supra may be included in a single vector with the TRAPsequence. Where this is not desired, then an expression system may beprovided, where two or more separate vectors are used where each codingsequence is operably linked to a promoter. Again, the promoter may be astrong or differential promoter. Co-transfection is a well knowntechnique, and the artisan in this field is expected to have thistechnology available for utilization. The vectors may be constructed sothat they code for the TRA molecule directly. rather than the TRAPmolecule. This eliminates the need for post-translational processing.

As the foregoing discussion makes clear, the sequences code for “tumorrejection antigen precursors” (“TRAPs”) which, in turn, are processedinto tumor rejection antigens (“TRAs”). Isolated forms of both of thesecategories are described herein, including specific examples of each.Perhaps their most noteworthy aspect is as vaccines for treating variouscancerous conditions. The evidence points to presentation of TRAs ontumor cells, followed by the development of an immune response anddeletion of the cells. The examples show that when various TRAs areadministered to cells, a CTL response is mounted and presenting cellsare deleted. This is behavior characteristic of vaccines, and henceTRAPs, which are processed into TRAs, and the TRAs themselves may beused, either alone or in pharmaceutically appropriate compositions, asvaccines. Similarly, presenting cells may be used in the same manner,either alone or as combined with ingredients to yield pharmaceuticalcompositions. Additional materials which may be used as vaccines includeisolated cells which present the TRA molecule on their surface, as wellas TRAP fragments, mutated viruses, especially etiolated forms, andtransfected bacteria. “Fragments” as used herein refers to peptideswhich are smaller than the TRAP, but which possess the propertiesrequired of a vaccine, as discussed supra. Another vaccine comprises orconsists of complexes of TRA and HLA molecule. Vaccines of the typedescribed herein may be used preventively, i.e., via administration to asubject in an amount sufficient to prevent onset of a cancerouscondition.

The generation of an immune response, be it T-cell or B-cell related, ischaracteristic of the effect of the presented tumor rejection antigen.With respect to the B-cell response, this involves, inter alia, thegeneration of antibodies to the TRA, i.e., which specifically bindthereto. In addition, the TRAP molecules are of sufficient size torender them immunogenic, and antibodies which specifically bind theretoare a part of this invention. These antibodies may be polyclonal ormonoclonal, the latter being prepared by any of the well recognizedmethodologies for their preparation which need not be repeated here. Forexample, mAbs may be prepared using an animal model, e.g., a Balb/Cmouse or in a test tube, using, e.g., EBV transformants. In addition,antiserum may be isolated from a subject afflicted with a cancerouscondition where certain cells present a TRA. Such antibodies may also begenerated to epitopes defined by the interaction of TRA and HLA/MHCmolecules.

Review of the foregoing disclosure will show that there are a number offacets to the system which may be referred to as “tumor rejectionantigen presentation and recognition”. Recognition of these phenomenahas diagnostic consequences. For example, the existence of specific CTLclones, or antibodies to the TRA makes it possible to diagnose ormonitor cancerous conditions (explained infra), by monitoring the CTLsin a sample from a subject, binding of antibodies to TRAs, or theactivity of anti-TRA CTLs in connection with subject samples. Similarly,the expression of nucleic acid molecules for TRAPs can be monitored viaamplification (e.g., “polymerase chain reaction”), anti-sensehybridization, probe technologies, and so forth. Various subjectsamples, including body fluids (blood, serum, and other exudates, e.g.),tissues and tumors may be so assayed.

A particular manner of diagnosis is to use an adaptation of the standard“tuberculin test” currently used for diagnosis of tuberculosis. Thisstandard skin test administers a stable form of “purified proteinderivative” or “PPD” as a diagnostic aid. In a parallel fashion, TRAs inaccordance with this invention may be used in such a skin test as adiagnostic aid or monitoring method.

The term “cancerous condition” is used herein to embrace allphysiological events that commence with the initiation of the cancer andresult in final clinical manifestation. Tumors do not spring up “abinitio” as visible tumors; rather there are various events associatedwith the transformation of a normal cell to malignancy, followed bydevelopment of a growth of biomass, such as a tumor, metastasis, etc. Inaddition, remission may be conceived of as part of “a cancerouscondition” as tumors seldom spontaneously disappear. The diagnosticaspects of this invention include all events involved in carcinogenesis,from the first transformation to malignancy of a single cell, throughtumor development and metastasis, as well as remission. All are embracedherein.

Where “subject” is used, the term embraces any species which can beafflicted with a cancerous condition. This includes humans andnon-humans, such as domesticated animals, breeding stock, and so forth.

There are therapeutic aspects of this invention as well. The efficacy ofadministration of effective amounts of TRAPs and TRAs as vaccines hasalready been discussed supra. Similarly, one may develop the specificCTLs in vitro and then administer these to the subject. Antibodies maybe administered, either polyclonal or monoclonal, which specificallybind to cells presenting the TRA of interest. These antibodies may becoupled to specific antitumor agents, including, but not being limitedto, methotrexate radio-iodinated compounds, toxins such as ricin, othercytostatic or cytolytic drugs, and so forth. Thus, “targeted” antibodytherapy is included herein, as is the application of deletion of thecancerous cells by the use of CTLs.

The data from examples 37-40 show that a tumor rejection antigen derivedfrom MAGE-3 is presented by HLA-A1 molecules. As such, in addition tothe nucleic acid molecules coding for this TRAP, the TRAP itself ascoded for by the sequences, vectors, cell lines, etcetera whichincorporate this nucleic acid molecule, the invention also encompassescombination of the molecules coding for the MAGE-3 TRAP and HLA-A1.Thus, co-transfectants, vectors containing coding sequences for both,expression systems such as kits, or separate vectors, and so forth, areall embraced by the invention. Similarly, the vaccines discussed supracan be made by incorporating the TRAP from MAGE-3 and an adjuvant.

It is to be understood that a given TRAP may yield more than one TRA. Inthe case of MAGE-3, it has been shown that antigen D, as the term isused herein, derives therefrom, and one aspect of the invention is thisisolated tumor rejection antigen. Another is isolated complexes of theTRA and its presenting molecule, i.e., EHLA-A1.

The identification of MAGE-3 derived TRAs as being presented by HLA-A1molecules suggests various therapeutic and diagnostic approaches. In atherapeutic context, e.g., the treatment of a disorder characterized byMAGE-3 expression may be treated in a number of ways, “disorder” beingused to refer to any pathological condition where MAGE-3 TRAP isexpressed, such as cancer (e.g., melanoma).

Therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, leading to lysis of TRApresenting cells, such as HLA-A1 cells. One such approach is theadministration of CTLs specific to the complex to a subject withabnormal cells of the phenotype at issue. it is within the skill of theartisan to develop such CTLs in vitro. Specifically, a sample of cells,such as blood cells, are contacted to a cell presenting the complex andcapable of provoking a specific CTL to proliferate. The target cell canbe a transfectant, such as a COS cell of the type described supra. Thesetransfectants present the desired complex on their surface and, whencombined with a CTL of interest, stimulate its proliferation. COS cells,such as those used herein are widely available, as are other suitablehost cells.

To detail the therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol. 136(5): 1917 (1986); Reddel et al., Science 257:238 (Jul. 10, 1992); Lynch et al., Eur. J. Immunol. 21: 1403-1410(1991); Kast et al., Cell 59: 603-614 (Nov. 17, 1989)), cells presentingthe desired complex are combined with CTLs leading to proliferation ofthe CTLs specific thereto. The proliferated CTLs are then administeredto a subject with a cellular abnormality which is characterized bycertain of the abnormal cells presenting the particular complex. TheCTLs then lyse the abnormal cells, thereby achieving the desiredtherapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the HLA/TRA complex. This can be determined veryeasily, as the art is very familiar with methods for identifying cellswhich present a particular HLA molecule, as well as how to identifycells expressing DNA containing the indicated sequences. Once isolated,such cells can be used with a sample of a subject's abnormal cells todetermine lysis in vitro. If lysis is observed, then the use of specificCTLs in such a therapy may alleviate the condition associated with theabnormal cells. A less involved methodology examines the abnormal cellsfor HLA phenotyping, using standard assays, and determines expressionvia amplification using, e.g., PCR.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach, i.e., the use of non-proliferativecells expressing the complex, has been elaborated upon supra. The cellsused in this approach may be those that normally express the complex,such as irradiated melanoma cells or cells transfected with one or bothof the genes necessary for presentation of the complex. Chen et al.,Proc. Natl. Acad. Sci. USA 88: 110-114 (January, 1991) exemplifies thisapproach, showing the use of transfected cells expressing HPVE7 peptidesin a therapeutic regime. Various cell types may be used. Similarly,vectors carrying one or both of the genes of interest may be used. Viralor bacterial vectors are especially preferred. In these systems, thegene of interest is carried by, e.g., a Vaccinia virus or the bacteriaBCG, and the materials de facto “infect” host cells. The cells whichresult present the complex of interest, and are recognized by autologousCTLs, which then proliferate. A similar effect can be achieved bycombining the tumor rejection antigen or the precursor itself with anadjuvant to facilitate incorporation into HLA-A1 presenting cells whichpresent the HLA molecule of interest. The TRAP is processed to yield thepeptide partner of the HLA molecule while the TRA is presented withoutthe need for further processing. Thus, one may treat disorders where aMAGE-3 derived TRA is presented by HLA-A1 molecules, or by any HLAmolecule.

In a diagnostic context, one may determine a disorder, as the term isused herein, by assaying for expression of the TRAP. This can be donedirectly (via, e.g., a PCR assay for TRAP sequences), or indirectly, viaassaying for a MAGE-3 derived TRA, as the TRA's presence means that theTRAP is or was expressed.

It will be noted that two nucleic acid molecules are presented herein,i.e., MAGE-3and MAGE-31, each of which code for TRAP MAGE-3. It is to beunderstood that when the epxression “nucleic acid molecule which codesfor MAGE-3 TRAP” is used, all molecules are covered which yield thismolecule upon expression. Any number of variations, such as thoseshowing codon degeneracy within the coding region, or variation withinthe introns, are covered by the invention.

The terms and expressions which have been employed are used as terms ofdescription and not of limitation, and there is no intention in the useof such terms and expressions of excluding any equivalents of thefeatures shown and described or portions thereof, it being recognizedthat various modifications are possible within the scope of theinvention.

30 462 base pairs nucleic acid single linear genomic DNA 1 ACCACAGGAGAATGAAAAGA ACCCGGGACT CCCAAAGACG CTAGATGTGT GAAGATCCTG 60 ATCACTCATTGGGTGTCTGA GTTCTGCGAT ATTCATCCCT CAGCCAATGA GCTTACTGTT 120 CTCGTGGGGGGTTTGTGAGC CTTGGGTAGG AAGTTTTGCA AGTTCCGCCT ACAGCTCTAG 180 CTTGTGAATTTGTACCCTTT CACGTAAAAA AGTAGTCCAG AGTTTACTAC ACCCTCCCTC 240 CCCCCTCCCACCTCGTGCTG TGCTGAGTTT AGAAGTCTTC CTTATAGAAG TCTTCCGTAT 300 AGAACTCTTCCGGAGGAAGG AGGGAGGACC CCCCCCCTTT GCTCTCCCAG CATGCATTGT 360 GTCAACGCCATTGCACTGAG CTGGTCGAAG AAGTAAGCCG CTAGCTTGCG ACTCTACTCT 420 TATCTTAACTTAGCTCGGCT TCCTGCTGGT ACCCTTTGTG CC 462 675 base pairs nucleic acidsingle linear genomic DNA 2 ATG TCT GAT AAC AAG AAA CCA GAC AAA GCC CACAGT GGC TCA GGT GGT 48 Met Ser Asp Asn Lys Lys Pro Asp Lys Ala His SerGly Ser Gly Gly 5 10 15 GAC GGT GAT GGG AAT AGG TGC AAT TTA TTG CAC CGGTAC TCC CTG GAA 96 Asp Gly Asp Gly Asn Arg Cys Asn Leu Leu His Arg TyrSer Leu Glu 20 25 30 GAA ATT CTG CCT TAT CTA GGG TGG CTG GTC TTC GCT GTTGTC ACA ACA 144 Glu Ile Leu Pro Tyr Leu Gly Trp Leu Val Phe Ala Val ValThr Thr 35 40 45 AGT TTT CTG GCG CTC CAG ATG TTC ATA GAC GCC CTT TAT GAGGAG CAG 192 Ser Phe Leu Ala Leu Gln Met Phe Ile Asp Ala Leu Tyr Glu GluGln 50 55 60 TAT GAA AGG GAT GTG GCC TGG ATA GCC AGG CAA AGC AAG CGC ATGTCC 240 Tyr Glu Arg Asp Val Ala Trp Ile Ala Arg Gln Ser Lys Arg Met Ser65 70 75 80 TCT GTC GAT GAG GAT GAA GAC GAT GAG GAT GAT GAG GAT GAC TACTAC 288 Ser Val Asp Glu Asp Glu Asp Asp Glu Asp Asp Glu Asp Asp Tyr Tyr85 90 95 GAC GAC GAG GAC GAC GAC GAC GAT GCC TTC TAT GAT GAT GAG GAT GAT336 Asp Asp Glu Asp Asp Asp Asp Asp Ala Phe Tyr Asp Asp Glu Asp Asp 100105 110 GAG GAA GAA GAA TTG GAG AAC CTG ATG GAT GAT GAA TCA GAA GAT GAG384 Glu Glu Glu Glu Leu Glu Asn Leu Met Asp Asp Glu Ser Glu Asp Glu 115120 125 GCC GAA GAA GAG ATG AGC GTG GAA ATG GGT GCC GGA GCT GAG GAA ATG432 Ala Glu Glu Glu Met Ser Val Glu Met Gly Ala Gly Ala Glu Glu Met 130135 140 GGT GCT GGC GCT AAC TGT GCC TGT GTT CCT GGC CAT CAT TTA AGG AAG480 Gly Ala Gly Ala Asn Cys Ala Cys Val Pro Gly His His Leu Arg Lys 145150 155 160 AAT GAA GTG AAG TGT AGG ATG ATT TAT TTC TTC CAC GAC CCT AATTTC 528 Asn Glu Val Lys Cys Arg Met Ile Tyr Phe Phe His Asp Pro Asn Phe165 170 175 CTG GTG TCT ATA CCA GTG AAC CCT AAG GAA CAA ATG GAG TGT AGGTGT 576 Leu Val Ser Ile Pro Val Asn Pro Lys Glu Gln Met Glu Cys Arg Cys180 185 190 GAA AAT GCT GAT GAA GAG GTT GCA ATG GAA GAG GAA GAA GAA GAAGAG 624 Glu Asn Ala Asp Glu Glu Val Ala Met Glu Glu Glu Glu Glu Glu Glu195 200 205 GAG GAG GAG GAG GAA GAG GAA ATG GGA AAC CCG GAT GGC TTC TCACCT 672 Glu Glu Glu Glu Glu Glu Glu Met Gly Asn Pro Asp Gly Phe Ser Pro210 215 220 TAG 675 228 base pairs nucleic acid single linear genomicDNA 3 GCATGCAGTT GCAAAGCCCA GAAGAAAGAA ATGGACAGCG GAAGAAGTGG TTGTTTTTTT60 TTCCCCTTCA TTAATTTTCT AGTTTTTAGT AATCCAGAAA ATTTGATTTT GTTCTAAAGT 120TCATTATGCA AAGATGTCAC CAACAGACTT CTGACTGCAT GGTGAACTTT CATATGATAC 180ATAGGATTAC ACTTGTACCT GTTAAAAATA AAAGTTTGAC TTGCATAC 228 1365 base pairsnucleic acid single linear genomic DNA 4 ACCACAGGAG AATGAAAAGAACCCGGGACT CCCAAAGACG CTAGATGTGT 50 GAAGATCCTG ATCACTCATT GGGTGTCTGAGTTCTGCGAT ATTCATCCCT 100 CAGCCAATGA GCTTACTGTT CTCGTGGGGG GTTTGTGAGCCTTGGGTAGG 150 AAGTTTTGCA AGTTCCGCCT ACAGCTCTAG CTTGTGAATT TGTACCCTTT200 CACGTAAAAA AGTAGTCCAG AGTTTACTAC ACCCTCCCTC CCCCCTCCCA 250CCTCGTGCTG TGCTGAGTTT AGAAGTCTTC CTTATAGAAG TCTTCCGTAT 300 AGAACTCTTCCGGAGGAAGG AGGGAGGACC CCCCCCCTTT GCTCTCCCAG 350 CATGCATTGT GTCAACGCCATTGCACTGAG CTGGTCGAAG AAGTAAGCCG 400 CTAGCTTGCG ACTCTACTCT TATCTTAACTTAGCTCGGCT TCCTGCTGGT 450 ACCCTTTGTG CC 462 ATG TCT GAT AAC AAG AAA CCAGAC AAA GCC CAC AGT GGC TCA 504 GGT GGT GAC GGT GAT GGG AAT AGG TGC AATTTA TTG CAC CGG 546 TAC TCC CTG GAA GAA ATT CTG CCT TAT CTA GGG TGG CTGGTC 588 TTC GCT GTT GTC ACA ACA AGT TTT CTG GCG CTC CAG ATG TTC 630 ATAGAC GCC CTT TAT GAG GAG CAG TAT GAA AGG GAT GTG GCC 672 TGG ATA GCC AGGCAA AGC AAG CGC ATG TCC TCT GTC GAT GAG 714 GAT GAA GAC GAT GAG GAT GATGAG GAT GAC TAC TAC GAC GAC 756 GAG GAC GAC GAC GAC GAT GCC TTC TAT GATGAT GAG GAT GAT 798 GAG GAA GAA GAA TTG GAG AAC CTG ATG GAT GAT GAA TCAGAA 840 GAT GAG GCC GAA GAA GAG ATG AGC GTG GAA ATG GGT GCC GGA 882 GCTGAG GAA ATG GGT GCT GGC GCT AAC TGT GCC TGT GTT CCT 924 GGC CAT CAT TTAAGG AAG AAT GAA GTG AAG TGT AGG ATG ATT 966 TAT TTC TTC CAC GAC CCT AATTTC CTG GTG TCT ATA CCA GTG 1008 AAC CCT AAG GAA CAA ATG GAG TGT AGG TGTGAA AAT GCT GAT 1050 GAA GAG GTT GCA ATG GAA GAG GAA GAA GAA GAA GAG GAGGAG 1092 GAG GAG GAA GAG GAA ATG GGA AAC CCG GAT GGC TTC TCA CCT 1134TAG 1137 GCATGCAGTT GCAAAGCCCA GAAGAAAGAA ATGGACAGCG GAAGAAGTGG 1187TTGTTTTTTT TTCCCCTTCA TTAATTTTCT AGTTTTTAGT AATCCAGAAA 1237 ATTTGATTTTGTTCTAAAGT TCATTATGCA AAGATGTCAC CAACAGACTT 1287 CTGACTGCAT GGTGAACTTTCATATGATAC ATAGGATTAC ACTTGTACCT 1337 GTTAAAAATA AAAGTTTGAC TTGCATAC1365 4698 base pairs nucleic acid single linear genomic DNA 5 ACCACAGGAGAATGAAAAGA ACCCGGGACT CCCAAAGACG CTAGATGTGT 50 GAAGATCCTG ATCACTCATTGGGTGTCTGA GTTCTGCGAT ATTCATCCCT 100 CAGCCAATGA GCTTACTGTT CTCGTGGGGGGTTTGTGAGC CTTGGGTAGG 150 AAGTTTTGCA AGTTCCGCCT ACAGCTCTAG CTTGTGAATTTGTACCCTTT 200 CACGTAAAAA AGTAGTCCAG AGTTTACTAC ACCCTCCCTC CCCCCTCCCA250 CCTCGTGCTG TGCTGAGTTT AGAAGTCTTC CTTATAGAAG TCTTCCGTAT 300AGAACTCTTC CGGAGGAAGG AGGGAGGACC CCCCCCCTTT GCTCTCCCAG 350 CATGCATTGTGTCAACGCCA TTGCACTGAG CTGGTCGAAG AAGTAAGCCG 400 CTAGCTTGCG ACTCTACTCTTATCTTAACT TAGCTCGGCT TCCTGCTGGT 450 ACCCTTTGTG CC 462 ATG TCT GAT AACAAG AAA CCA GAC AAA GCC CAC AGT GGC TCA 504 GGT GGT GAC GGT GAT GGG AATAGG TGC AAT TTA TTG CAC CGG 546 TAC TCC CTG GAA GAA ATT CTG CCT TAT CTAGGG TGG CTG GTC 588 TTC GCT GTT GTC ACA ACA AGT TTT CTG GCG CTC CAG ATGTTC 630 ATA GAC GCC CTT TAT GAG GAG CAG TAT GAA AGG GAT GTG GCC 672 TGGATA GCC AGG CAA AGC AAG CGC ATG TCC TCT GTC GAT GAG 714 GAT GAA GAC GATGAG GAT GAT GAG GAT GAC TAC TAC GAC GAC 756 GAG GAC GAC GAC GAC GAT GCCTTC TAT GAT GAT GAG GAT GAT 798 GAG GAA GAA GAA TTG GAG AAC CTG ATG GATGAT GAA TCA GAA 840 GAT GAG GCC GAA GAA GAG ATG AGC GTG GAA ATG GGT GCCGGA 882 GCT GAG GAA ATG GGT GCT GGC GCT AAC TGT GCC T 916 GTGAGTAACCCGTGGTCTTT ACTCTAGATT CAGGTGGGGT GCATTCTTTA 966 CTCTTGCCCA CATCTGTAGTAAAGACCACA TTTTGGTTGG GGGTCATTGC 1016 TGGAGCCATT CCTGGCTCTC CTGTCCACGCCTATCCCCGC TCCTCCCATC 1066 CCCCACTCCT TGCTCCGCTC TCTTTCCTTT TCCCACCTTGCCTCTGGAGC 1116 TTCAGTCCAT CCTGCTCTGC TCCCTTTCCC CTTTGCTCTC CTTGCTCCCC1166 TCCCCCTCGG CTCAACTTTT CGTGCCTTCT GCTCTCTGAT CCCCACCCTC 1216TTCAGGCTTC CCCATTTGCT CCTCTCCCGA AACCCTCCCC TTCCTGTTCC 1266 CCTTTTCGCGCCTTTTCTTT CCTGCTCCCC TCCCCCTCCC TATTTACCTT 1316 TCACCAGCTT TGCTCTCCCTGCTCCCCTCC CCCTTTTGCA CCTTTTCTTT 1366 TCCTGCTCCC CTCCCCCTCC CCTCCCTGTTTACCCTTCAC CGCTTTTCCT 1416 CTACCTGCTT CCCTCCCCCT TGCTGCTCCC TCCCTATTTGCATTTTCGGG 1466 TGCTCCTCCC TCCCCCTCCC CCTCCCTCCC TATTTGCATT TTCGGGTGCT1516 CCTCCCTCCC CCTCCCCAGG CCTTTTTTTT TTTTTTTTTT TTTTTTTTTT 1566TTGGTTTTTC GAGACAGGGT TTCTCTTTGT ATCCCTGGCT GTCCTGGCAC 1616 TCACTCTGTAGACCAGGCTG GCCTCAAACT CAGAAATCTG CCTGCCTCTG 1666 CCTCCCAAAT GCTGGGATTAAAGGCTTGCA CCAGGACTGC CCCAGTGCAG 1716 GCCTTTCTTT TTTCTCCTCT CTGGTCTCCCTAATCCCTTT TCTGCATGTT 1766 AACTCCCCTT TTGGCACCTT TCCTTTACAG GACCCCCTCCCCCTCCCTGT 1816 TTCCCTTCCG GCACCCTTCC TAGCCCTGCT CTGTTCCCTC TCCCTGCTCC1866 CCTCCCCCTC TTTGCTCGAC TTTTAGCAGC CTTACCTCTC CCTGCTTTCT 1916GCCCCGTTCC CCTTTTTTGT GCCTTTCCTC CTGGCTCCCC TCCACCTTCC 1966 AGCTCACCTTTTTGTTTGTT TGGTTGTTTG GTTGTTTGGT TTGCTTTTTT 2016 TTTTTTTTTT GCACCTTGTTTTCCAAGATC CCCCTCCCCC TCCGGCTTCC 2066 CCTCTGTGTG CCTTTCCTGT TCCCTCCCCCTCGCTGGCTC CCCCTCCCTT 2116 TCTGCCTTTC CTGTCCCTGC TCCCTTCTCT GCTAACCTTTTAATGCCTTT 2166 CTTTTCTAGA CTCCCCCCTC CAGGCTTGCT GTTTGCTTCT GTGCACTTTT2216 CCTGACCCTG CTCCCCTTCC CCTCCCAGCT CCCCCCTCTT TTCCCACCTC 2266CCTTTCTCCA GCCTGTCACC CCTCCTTCTC TCCTCTCTGT TTCTCCCACT 2316 TCCTGCTTCCTTTACCCCTT CCCTCTCCCT ACTCTCCTCC CTGCCTGCTG 2366 GACTTCCTCT CCAGCCGCCCAGTTCCCTGC AGTCCTGGAG TCTTTCCTGC 2416 CTCTCTGTCC ATCACTTCCC CCTAGTTTCACTTCCCTTTC ACTCTCCCCT 2466 ATGTGTCTCT CTTCCTATCT ATCCCTTCCT TTCTGTCCCCTCTCCTCTGT 2516 CCATCACCTC TCTCCTCCCT TCCCTTTCCT CTCTCTTCCA TTTTCTTCCA2566 CCTGCTTCTT TACCCTGCCT CTCCCATTGC CCTCTTACCT TTATGCCCAT 2616TCCATGTCCC CTCTCAATTC CCTGTCCCAT TGTGCTCCCT CACATCTTCC 2666 ATTTCCCTCTTTCTCCCTTA GCCTCTTCTT CCTCTTCTCT TGTATCTCCC 2716 TTCCCTTTGC TTCTCCCTCCTCCTTTCCCC TTCCCCTATG CCCTCTACTC 2766 TACTTGATCT TCTCTCCTCT CCACATACCCTTTTTCCTTT CCACCCTGCC 2816 CTTTGTCCCC AGACCCTACA GTATCCTGTG CACAGGAAGTGGGAGGTGCC 2866 ATCAACAACA AGGAGGCAAG AAACAGAGCA AAATCCCAAA ATCAGCAGGA2916 AAGGCTGGAT GAAAATAAGG CCAGGTTCTG AGGACAGCTG GAATCTAGCC 2966AAGTGGCTCC TATAACCCTA AGTACCAAGG GAGAAAGTGA TGGTGAAGTT 3016 CTTGATCCTTGCTGCTTCTT TTACATATGT TGGCACATCT TTCTCAAATG 3066 CAGGCCATGC TCCATGCTTGGCGCTTGCTC AGCGTGGTTA AGTAATGGGA 3116 GAATCTGAAA ACTAGGGGCC AGTGGTTTGTTTTGGGGACA AATTAGCACG 3166 TAGTGATATT TCCCCCTAAA AATTATAACA AACAGATTCATGATTTGAGA 3216 TCCTTCTACA GGTGAGAAGT GGAAAAATTG TCACTATGAA GTTCTTTTTA3266 GGCTAAAGAT ACTTGGAACC ATAGAAGCGT TGTTAAAATA CTGCTTTCTT 3316TTGCTAAAAT ATTCTTTCTC ACATATTCAT ATTCTCCAG 3355 GT GTT CCT GGC CAT CATTTA AGG AAG AAT GAA GTG AAG TGT 3396 AGG ATG ATT TAT TTC TTC CAC GAC CCTAAT TTC CTG GTG TCT 3438 ATA CCA GTG AAC CCT AAG GAA CAA ATG GAG TGT AGGTGT GAA 3480 AAT GCT GAT GAA GAG GTT GCA ATG GAA GAG GAA GAA GAA GAA3522 GAG GAG GAG GAG GAG GAA GAG GAA ATG GGA AAC CCG GAT GGC 3564 TTCTCA CCT TAG 3576 GCATGCAGGT ACTGGCTTCA CTAACCAACC ATTCCTAACA TATGCCTGTA3626 GCTAAGAGCA TCTTTTTAAA AAATATTATT GGTAAACTAA ACAATTGTTA 3676TCTTTTTACA TTAATAAGTA TTAAATTAAT CCAGTATACA GTTTTAAGAA 3726 CCCTAAGTTAAACAGAAGTC AATGATGTCT AGATGCCTGT TCTTTAGATT 3776 GTAGTGAGAC TACTTACTACAGATGAGAAG TTGTTAGACT CGGGAGTAGA 3826 GACCAGTAAA AGATCATGCA GTGAAATGTGGCCATGGAAA TCGCATATTG 3876 TTCTTATAGT ACCTTTGAGA CAGCTGATAA CAGCTGACAAAAATAAGTGT 3926 TTCAAGAAAG ATCACACGCC ATGGTTCACA TGCAAATTAT TATTTTGTCG3976 TTCTGATTTT TTTCATTTCT AGACCTGTGG TTTTAAAGAG ATGAAAATCT 4026CTTAAAATTT CCTTCATCTT TAATTTTCCT TAACTTTAGT TTTTTTCACT 4076 TAGAATTCAATTCAAATTCT TAATTCAATC TTAATTTTTA GATTTCTTAA 4126 AATGTTTTTT AAAAAAAATGCAAATCTCAT TTTTAAGAGA TGAAAGCAGA 4176 GTAACTGGGG GGCTTAGGGA ATCTGTAGGGTTGCGGTATA GCAATAGGGA 4226 GTTCTGGTCT CTGAGAAGCA GTCAGAGAGA ATGGAAAACCAGGCCCTTGC 4276 CAGTAGGTTA GTGAGGTTGA TATGATCAGA TTATGGACAC TCTCCAAATC4326 ATAAATACTC TAACAGCTAA GGATCTCTGA GGGAAACACA ACAGGGAAAT 4376ATTTTAGTTT CTCCTTGAGA AACAATGACA AGACATAAAA TTGGCAAGAA 4426 AGTCAGGAGTGTATTCTAAT AAGTGTTGCT TATCTCTTAT TTTCTTCTAC 4476 AGTTGCAAAG CCCAGAAGAAAGAAATGGAC AGCGGAAGAA GTGGTTGTTT 4526 TTTTTTCCCC TTCATTAATT TTCTAGTTTTTAGTAATCCA GAAAATTTGA 4576 TTTTGTTCTA AAGTTCATTA TGCAAAGATG TCACCAACAGACTTCTGACT 4626 GCATGGTGAA CTTTCATATG ATACATAGGA TTACACTTGT ACCTGTTAAA4676 AATAAAAGTT TGACTTGCAT AC 4698 9 amino acids amino acid linearprotein 6 Leu Pro Tyr Leu Gly Trp Leu Val Phe 5 2419 base pairs nucleicacid single linear genomic DNA 7 GGATCCAGGC CCTGCCAGGA AAAATATAAGGGCCCTGCGT GAGAACAGAG 50 GGGGTCATCC ACTGCATGAG AGTGGGGATG TCACAGAGTCCAGCCCACCC 100 TCCTGGTAGC ACTGAGAAGC CAGGGCTGTG CTTGCGGTCT GCACCCTGAG150 GGCCCGTGGA TTCCTCTTCC TGGAGCTCCA GGAACCAGGC AGTGAGGCCT 200TGGTCTGAGA CAGTATCCTC AGGTCACAGA GCAGAGGATG CACAGGGTGT 250 GCCAGCAGTGAATGTTTGCC CTGAATGCAC ACCAAGGGCC CCACCTGCCA 300 CAGGACACAT AGGACTCCACAGAGTCTGGC CTCACCTCCC TACTGTCAGT 350 CCTGTAGAAT CGACCTCTGC TGGCCGGCTGTACCCTGAGT ACCCTCTCAC 400 TTCCTCCTTC AGGTTTTCAG GGGACAGGCC AACCCAGAGGACAGGATTCC 450 CTGGAGGCCA CAGAGGAGCA CCAAGGAGAA GATCTGTAAG TAGGCCTTTG500 TTAGAGTCTC CAAGGTTCAG TTCTCAGCTG AGGCCTCTCA CACACTCCCT 550CTCTCCCCAG GCCTGTGGGT CTTCATTGCC CAGCTCCTGC CCACACTCCT 600 GCCTGCTGCCCTGACGAGAG TCATCATGTC TCTTGAGCAG AGGAGTCTGC 650 ACTGCAAGCC TGAGGAAGCCCTTGAGGCCC AACAAGAGGC CCTGGGCCTG 700 GTGTGTGTGC AGGCTGCCAC CTCCTCCTCCTCTCCTCTGG TCCTGGGCAC 750 CCTGGAGGAG GTGCCCACTG CTGGGTCAAC AGATCCTCCCCAGAGTCCTC 800 AGGGAGCCTC CGCCTTTCCC ACTACCATCA ACTTCACTCG ACAGAGGCAA850 CCCAGTGAGG GTTCCAGCAG CCGTGAAGAG GAGGGGCCAA GCACCTCTTG 900TATCCTGGAG TCCTTGTTCC GAGCAGTAAT CACTAAGAAG GTGGCTGATT 950 TGGTTGGTTTTCTGCTCCTC AAATATCGAG CCAGGGAGCC AGTCACAAAG 1000 GCAGAAATGC TGGAGAGTGTCATCAAAAAT TACAAGCACT GTTTTCCTGA 1050 GATCTTCGGC AAAGCCTCTG AGTCCTTGCAGCTGGTCTTT GGCATTGACG 1100 TGAAGGAAGC AGACCCCACC GGCCACTCCT ATGTCCTTGTCACCTGCCTA 1150 GGTCTCTCCT ATGATGGCCT GCTGGGTGAT AATCAGATCA TGCCCAAGAC1200 AGGCTTCCTG ATAATTGTCC TGGTCATGAT TGCAATGGAG GGCGGCCATG 1250CTCCTGAGGA GGAAATCTGG GAGGAGCTGA GTGTGATGGA GGTGTATGAT 1300 GGGAGGGAGCACAGTGCCTA TGGGGAGCCC AGGAAGCTGC TCACCCAAGA 1350 TTTGGTGCAG GAAAAGTACCTGGAGTACGG CAGGTGCCGG ACAGTGATCC 1400 CGCACGCTAT GAGTTCCTGT GGGGTCCAAGGGCCCTCGCT GAAACCAGCT 1450 ATGTGAAAGT CCTTGAGTAT GTGATCAAGG TCAGTGCAAGAGTTCGCTTT 1500 TTCTTCCCAT CCCTGCGTGA AGCAGCTTTG AGAGAGGAGG AAGAGGGAGT1550 CTGAGCATGA GTTGCAGCCA AGGCCAGTGG GAGGGGGACT GGGCCAGTGC 1600ACCTTCCAGG GCCGCGTCCA GCAGCTTCCC CTGCCTCGTG TGACATGAGG 1650 CCCATTCTTCACTCTGAAGA GAGCGGTCAG TGTTCTCAGT AGTAGGTTTC 1700 TGTTCTATTG GGTGACTTGGAGATTTATCT TTGTTCTCTT TTGGAATTGT 1750 TCAAATGTTT TTTTTTAAGG GATGGTTGAATGAACTTCAG CATCCAAGTT 1800 TATGAATGAC AGCAGTCACA CAGTTCTGTG TATATAGTTTAAGGGTAAGA 1850 GTCTTGTGTT TTATTCAGAT TGGGAAATCC ATTCTATTTT GTGAATTGGG1900 ATAATAACAG CAGTGGAATA AGTACTTAGA AATGTGAAAA ATGAGCAGTA 1950AAATAGATGA GATAAAGAAC TAAAGAAATT AAGAGATAGT CAATTCTTGC 2000 CTTATACCTCAGTCTATTCT GTAAAATTTT TAAAGATATA TGCATACCTG 2050 GATTTCCTTG GCTTCTTTGAGAATGTAAGA GAAATTAAAT CTGAATAAAG 2100 AATTCTTCCT GTTCACTGGC TCTTTTCTTCTCCATGCACT GAGCATCTGC 2150 TTTTTGGAAG GCCCTGGGTT AGTAGTGGAG ATGCTAAGGTAAGCCAGACT 2200 CATACCCACC CATAGGGTCG TAGAGTCTAG GAGCTGCAGT CACGTAATCG2250 AGGTGGCAAG ATGTCCTCTA AAGATGTAGG GAAAAGTGAG AGAGGGGTGA 2300GGGTGTGGGG CTCCGGGTGA GAGTGGTGGA GTGTCAATGC CCTGAGCTGG 2350 GGCATTTTGGGCTTTGGGAA ACTGCAGTTC CTTCTGGGGG AGCTGATTGT 2400 AATGATCTTG GGTGGATCC2419 5674 base pairs nucleic acid single linear genomic DNA MAGE-1 gene8 CCCGGGGCAC CACTGGCATC CCTCCCCCTA CCACCCCCAA TCCCTCCCTT 50 TACGCCACCCATCCAAACAT CTTCACGCTC ACCCCCAGCC CAAGCCAGGC 100 AGAATCCGGT TCCACCCCTGCTCTCAACCC AGGGAAGCCC AGGTGCCCAG 150 ATGTGACGCC ACTGACTTGA GCATTAGTGGTTAGAGAGAA GCGAGGTTTT 200 CGGTCTGAGG GGCGGCTTGA GATCGGTGGA GGGAAGCGGGCCCAGCTCTG 250 TAAGGAGGCA AGGTGACATG CTGAGGGAGG ACTGAGGACC CACTTACCCC300 AGATAGAGGA CCCCAAATAA TCCCTTCATG CCAGTCCTGG ACCATCTGGT 350GGTGGACTTC TCAGGCTGGG CCACCCCCAG CCCCCTTGCT GCTTAAACCA 400 CTGGGGACTCGAAGTCAGAG CTCCGTGTGA TCAGGGAAGG GCTGCTTAGG 450 AGAGGGCAGC GTCCAGGCTCTGCCAGACAT CATGCTCAGG ATTCTCAAGG 500 AGGGCTGAGG GTCCCTAAGA CCCCACTCCCGTGACCCAAC CCCCACTCCA 550 ATGCTCACTC CCGTGACCCA ACCCCCTCTT CATTGTCATTCCAACCCCCA 600 CCCCACATCC CCCACCCCAT CCCTCAACCC TGATGCCCAT CCGCCCAGCC650 ATTCCACCCT CACCCCCACC CCCACCCCCA CGCCCACTCC CACCCCCACC 700CAGGCAGGAT CCGGTTCCCG CCAGGAAACA TCCGGGTGCC CGGATGTGAC 750 GCCACTGACTTGCGCATTGT GGGGCAGAGA GAAGCGAGGT TTCCATTCTG 800 AGGGACGGCG TAGAGTTCGGCCGAAGGAAC CTGACCCAGG CTCTGTGAGG 850 AGGCAAGGTG AGAGGCTGAG GGAGGACTGAGGACCCCGCC ACTCCAAATA 900 GAGAGCCCCA AATATTCCAG CCCCGCCCTT GCTGCCAGCCCTGGCCCACC 950 CGCGGGAAGA CGTCTCAGCC TGGGCTGCCC CCAGACCCCT GCTCCAAAAG1000 CCTTGAGAGA CACCAGGTTC TTCTCCCCAA GCTCTGGAAT CAGAGGTTGC 1050TGTGACCAGG GCAGGACTGG TTAGGAGAGG GCAGGGCACA GGCTCTGCCA 1100 GGCATCAAGATCAGCACCCA AGAGGGAGGG CTGTGGGCCC CCAAGACTGC 1150 ACTCCAATCC CCACTCCCACCCCATTCGCA TTCCCATTCC CCACCCAACC 1200 CCCATCTCCT CAGCTACACC TCCACCCCCATCCCTACTCC TACTCCGTCA 1250 CCTGACCACC ACCCTCCAGC CCCAGCACCA GCCCCAACCCTTCTGCCACC 1300 TCACCCTCAC TGCCCCCAAC CCCACCCTCA TCTCTCTCAT GTGCCCCACT1350 CCCATCGCCT CCCCCATTCT GGCAGAATCC GGTTTGCCCC TGCTCTCAAC 1400CCAGGGAAGC CCTGGTAGGC CCGATGTGAA ACCACTGACT TGAACCTCAC 1450 AGATCTGAGAGAAGCCAGGT TCATTTAATG GTTCTGAGGG GCGGCTTGAG 1500 ATCCACTGAG GGGAGTGGTTTTAGGCTCTG TGAGGAGGCA AGGTGAGATG 1550 CTGAGGGAGG ACTGAGGAGG CACACACCCCAGGTAGATGG CCCCAAAATG 1600 ATCCAGTACC ACCCCTGCTG CCAGCCCTGG ACCACCCGGCCAGGACAGAT 1650 GTCTCAGCTG GACCACCCCC CGTCCCGTCC CACTGCCACT TAACCCACAG1700 GGCAATCTGT AGTCATAGCT TATGTGACCG GGGCAGGGTT GGTCAGGAGA 1750GGCAGGGCCC AGGCATCAAG GTCCAGCATC CGCCCGGCAT TAGGGTCAGG 1800 ACCCTGGGAGGGAACTGAGG GTTCCCCACC CACACCTGTC TCCTCATCTC 1850 CACCGCCACC CCACTCACATTCCCATACCT ACCCCCTACC CCCAACCTCA 1900 TCTTGTCAGA ATCCCTGCTG TCAACCCACGGAAGCCACGG GAATGGCGGC 1950 CAGGCACTCG GATCTTGACG TCCCCATCCA GGGTCTGATGGAGGGAAGGG 2000 GCTTGAACAG GGCCTCAGGG GAGCAGAGGG AGGGCCCTAC TGCGAGATGA2050 GGGAGGCCTC AGAGGACCCA GCACCCTAGG ACACCGCACC CCTGTCTGAG 2100ACTGAGGCTG CCACTTCTGG CCTCAAGAAT CAGAACGATG GGGACTCAGA 2150 TTGCATGGGGGTGGGACCCA GGCCTGCAAG GCTTACGCGG AGGAAGAGGA 2200 GGGAGGACTC AGGGGACCTTGGAATCCAGA TCAGTGTGGA CCTCGGCCCT 2250 GAGAGGTCCA GGGCACGGTG GCCACATATGGCCCATATTT CCTGCATCTT 2300 TGAGGTGACA GGACAGAGCT GTGGTCTGAG AAGTGGGGCCTCAGGTCAAC 2350 AGAGGGAGGA GTTCCAGGAT CCATATGGCC CAAGATGTGC CCCCTTCATG2400 AGGACTGGGG ATATCCCCGG CTCAGAAAGA AGGGACTCCA CACAGTCTGG 2450CTGTCCCCTT TTAGTAGCTC TAGGGGGACC AGATCAGGGA TGGCGGTATG 2500 TTCCATTCTCACTTGTACCA CAGGCAGGAA GTTGGGGGGC CCTCAGGGAG 2550 ATGGGGTCTT GGGGTAAAGGGGGGATGTCT ACTCATGTCA GGGAATTGGG 2600 GGTTGAGGAA GCACAGGCGC TGGCAGGAATAAAGATGAGT GAGACAGACA 2650 AGGCTATTGG AATCCACACC CCAGAACCAA AGGGGTCAGCCCTGGACACC 2700 TCACCCAGGA TGTGGCTTCT TTTTCACTCC TGTTTCCAGA TCTGGGGCAG2750 GTGAGGACCT CATTCTCAGA GGGTGACTCA GGTCAACGTA GGGACCCCCA 2800TCTGGTCTAA AGACAGAGCG GTCCCAGGAT CTGCCATGCG TTCGGGTGAG 2850 GAACATGAGGGAGGACTGAG GGTACCCCAG GACCAGAACA CTGAGGGAGA 2900 CTGCACAGAA ATCAGCCCTGCCCCTGCTGT CACCCCAGAG AGCATGGGCT 2950 GGGCCGTCTG CCGAGGTCCT TCCGTTATCCTGGGATCATT GATGTCAGGG 3000 ACGGGGAGGC CTTGGTCTGA GAAGGCTGCG CTCAGGTCAGTAGAGGGAGC 3050 GTCCCAGGCC CTGCCAGGAG TCAAGGTGAG GACCAAGCGG GCACCTCACC3100 CAGGACACAT TAATTCCAAT GAATTTTGAT ATCTCTTGCT GCCCTTCCCC 3150AAGGACCTAG GCACGTGTGG CCAGATGTTT GTCCCCTCCT GTCCTTCCAT 3200 TCCTTATCATGGATGTGAAC TCTTGATTTG GATTTCTCAG ACCAGCAAAA 3250 GGGCAGGATC CAGGCCCTGCCAGGAAAAAT ATAAGGGCCC TGCGTGAGAA 3300 CAGAGGGGGT CATCCACTGC ATGAGAGTGGGGATGTCACA GAGTCCAGCC 3350 CACCCTCCTG GTAGCACTGA GAAGCCAGGG CTGTGCTTGCGGTCTGCACC 3400 CTGAGGGCCC GTGGATTCCT CTTCCTGGAG CTCCAGGAAC CAGGCAGTGA3450 GGCCTTGGTC TGAGACAGTA TCCTCAGGTC ACAGAGCAGA GGATGCACAG 3500GGTGTGCCAG CAGTGAATGT TTGCCCTGAA TGCACACCAA GGGCCCCACC 3550 TGCCACAGGACACATAGGAC TCCACAGAGT CTGGCCTCAC CTCCCTACTG 3600 TCAGTCCTGT AGAATCGACCTCTGCTGGCC GGCTGTACCC TGAGTACCCT 3650 CTCACTTCCT CCTTCAGGTT TTCAGGGGACAGGCCAACCC AGAGGACAGG 3700 ATTCCCTGGA GGCCACAGAG GAGCACCAAG GAGAAGATCTGTAAGTAGGC 3750 CTTTGTTAGA GTCTCCAAGG TTCAGTTCTC AGCTGAGGCC TCTCACACAC3800 TCCCTCTCTC CCCAGGCCTG TGGGTCTTCA TTGCCCAGCT CCTGCCCACA 3850CTCCTGCCTG CTGCCCTGAC GAGAGTCATC 3880 ATG TCT CTT GAG CAG AGG AGT CTGCAC TGC AAG CCT GAG GAA 3922 GCC CTT GAG GCC CAA CAA GAG GCC CTG GGC CTGGTG TGT GTG 3964 CAG GCT GCC ACC TCC TCC TCC TCT CCT CTG GTC CTG GGC ACC4006 CTG GAG GAG GTG CCC ACT GCT GGG TCA ACA GAT CCT CCC CAG 4048 AGTCCT CAG GGA GCC TCC GCC TTT CCC ACT ACC ATC AAC TTC 4090 ACT CGA CAG AGGCAA CCC AGT GAG GGT TCC AGC AGC CGT GAA 4132 GAG GAG GGG CCA AGC ACC TCTTGT ATC CTG GAG TCC TTG TTC 4174 CGA GCA GTA ATC ACT AAG AAG GTG GCT GATTTG GTT GGT TTT 4216 CTG CTC CTC AAA TAT CGA GCC AGG GAG CCA GTC ACA AAGGCA 4258 GAA ATG CTG GAG AGT GTC ATC AAA AAT TAC AAG CAC TGT TTT 4300CCT GAG ATC TTC GGC AAA GCC TCT GAG TCC TTG CAG CTG GTC 4342 TTT GGC ATTGAC GTG AAG GAA GCA GAC CCC ACC GGC CAC TCC 4384 TAT GTC CTT GTC ACC TGCCTA GGT CTC TCC TAT GAT GGC CTG 4426 CTG GGT GAT AAT CAG ATC ATG CCC AAGACA GGC TTC CTG ATA 4468 ATT GTC CTG GTC ATG ATT GCA ATG GAG GGC GGC CATGCT CCT 4510 GAG GAG GAA ATC TGG GAG GAG CTG AGT GTG ATG GAG GTG TAT4552 GAT GGG AGG GAG CAC AGT GCC TAT GGG GAG CCC AGG AAG CTG 4594 CTCACC CAA GAT TTG GTG CAG GAA AAG TAC CTG GAG TAC GGC 4636 AGG TGC CGG ACAGTG ATC CCG CAC GCT ATG AGT TCC TGT GGG 4678 GTC CAA GGG CCC TCG CTG AAACCA GCT ATG TGA 4711 AAGTCCTTGA GTATGTGATC AAGGTCAGTG CAAGAGTTC 4750GCTTTTTCTT CCCATCCCTG CGTGAAGCAG CTTTGAGAGA GGAGGAAGAG 4800 GGAGTCTGAGCATGAGTTGC AGCCAAGGCC AGTGGGAGGG GGACTGGGCC 4850 AGTGCACCTT CCAGGGCCGCGTCCAGCAGC TTCCCCTGCC TCGTGTGACA 4900 TGAGGCCCAT TCTTCACTCT GAAGAGAGCGGTCAGTGTTC TCAGTAGTAG 4950 GTTTCTGTTC TATTGGGTGA CTTGGAGATT TATCTTTGTTCTCTTTTGGA 5000 ATTGTTCAAA TGTTTTTTTT TAAGGGATGG TTGAATGAAC TTCAGCATCC5050 AAGTTTATGA ATGACAGCAG TCACACAGTT CTGTGTATAT AGTTTAAGGG 5100TAAGAGTCTT GTGTTTTATT CAGATTGGGA AATCCATTCT ATTTTGTGAA 5150 TTGGGATAATAACAGCAGTG GAATAAGTAC TTAGAAATGT GAAAAATGAG 5200 CAGTAAAATA GATGAGATAAAGAACTAAAG AAATTAAGAG ATAGTCAATT 5250 CTTGCCTTAT ACCTCAGTCT ATTCTGTAAAATTTTTAAAG ATATATGCAT 5300 ACCTGGATTT CCTTGGCTTC TTTGAGAATG TAAGAGAAATTAAATCTGAA 5350 TAAAGAATTC TTCCTGTTCA CTGGCTCTTT TCTTCTCCAT GCACTGAGCA5400 TCTGCTTTTT GGAAGGCCCT GGGTTAGTAG TGGAGATGCT AAGGTAAGCC 5450AGACTCATAC CCACCCATAG GGTCGTAGAG TCTAGGAGCT GCAGTCACGT 5500 AATCGAGGTGGCAAGATGTC CTCTAAAGAT GTAGGGAAAA GTGAGAGAGG 5550 GGTGAGGGTG TGGGGCTCCGGGTGAGAGTG GTGGAGTGTC AATGCCCTGA 5600 GCTGGGGCAT TTTGGGCTTT GGGAAACTGCAGTTCCTTCT GGGGGAGCTG 5650 ATTGTAATGA TCTTGGGTGG ATCC 5674 4157 basepairs nucleic acid single linear genomic DNA MAGE-2 gene 9 CCCATCCAGATCCCCATCCG GGCAGAATCC GGTTCCACCC TTGCCGTGAA 50 CCCAGGGAAG TCACGGGCCCGGATGTGACG CCACTGACTT GCACATTGGA 100 GGTCAGAGGA CAGCGAGATT CTCGCCCTGAGCAACGGCCT GACGTCGGCG 150 GAGGGAAGCA GGCGCAGGCT CCGTGAGGAG GCAAGGTAAGACGCCGAGGG 200 AGGACTGAGG CGGGCCTCAC CCCAGACAGA GGGCCCCCAA TTAATCCAGC250 GCTGCCTCTG CTGCCGGGCC TGGACCACCC TGCAGGGGAA GACTTCTCAG 300GCTCAGTCGC CACCACCTCA CCCCGCCACC CCCCGCCGCT TTAACCGCAG 350 GGAACTCTGGCGTAAGAGCT TTGTGTGACC AGGGCAGGGC TGGTTAGAAG 400 TGCTCAGGGC CCAGACTCAGCCAGGAATCA AGGTCAGGAC CCCAAGAGGG 450 GACTGAGGGC AACCCACCCC CTACCCTCACTACCAATCCC ATCCCCCAAC 500 ACCAACCCCA CCCCCATCCC TCAAACACCA ACCCCACCCCCAAACCCCAT 550 TCCCATCTCC TCCCCCACCA CCATCCTGGC AGAATCCGGC TTTGCCCCTG600 CAATCAACCC ACGGAAGCTC CGGGAATGGC GGCCAAGCAC GCGGATCCTG 650ACGTTCACAT GTACGGCTAA GGGAGGGAAG GGGTTGGGTC TCGTGAGTAT 700 GGCCTTTGGGATGCAGAGGA AGGGCCCAGG CCTCCTGGAA GACAGTGGAG 750 TCCTTAGGGG ACCCAGCATGCCAGGACAGG GGGCCCACTG TACCCCTGTC 800 TCAAACTGAG CCACCTTTTC ATTCAGCCGAGGGAATCCTA GGGATGCAGA 850 CCCACTTCAG GGGGTTGGGG CCCAGCCTGC GAGGAGTCAAGGGGAGGAAG 900 AAGAGGGAGG ACTGAGGGGA CCTTGGAGTC CAGATCAGTG GCAACCTTGG950 GCTGGGGGAT CCTGGGCACA GTGGCCGAAT GTGCCCCGTG CTCATTGCAC 1000CTTCAGGGTG ACAGAGAGTT GAGGGCTGTG GTCTGAGGGC TGGGACTTCA 1050 GGTCAGCAGAGGGAGGAATC CCAGGATCTG CCGGACCCAA GGTGTGCCCC 1100 CTTCATGAGG ACTCCCCATACCCCCGGCCC AGAAAGAAGG GATGCCACAG 1150 AGTCTGGAAG TAAATTGTTC TTAGCTCTGGGGGAACCTGA TCAGGGATGG 1200 CCCTAAGTGA CAATCTCATT TGTACCACAG GCAGGAGGTTGGGGAACCCT 1250 CAGGGAGATA AGGTGTTGGT GTAAAGAGGA GCTGTCTGCT CATTTCAGGG1300 GGTTCCCCCT TGAGAAAGGG CAGTCCCTGG CAGGAGTAAA GATGAGTAAC 1350CCACAGGAGG CCATCATAAC GTTCACCCTA GAACCAAAGG GGTCAGCCCT 1400 GGACAACGCACGTGGGGTAA CAGGATGTGG CCCCTCCTCA CTTGTCTTTC 1450 CAGATCTCAG GGAGTTGATGACCTTGTTTT CAGAAGGTGA CTCAGTCAAC 1500 ACAGGGGCCC CTCTGGTCGA CAGATGCAGTGGTTCTAGGA TCTGCCAAGC 1550 ATCCAGGTGG AGAGCCTGAG GTAGGATTGA GGGTACCCCTGGGCCAGAAT 1600 GCAGCAAGGG GGCCCCATAG AAATCTGCCC TGCCCCTGCG GTTACTTCAG1650 AGACCCTGGG CAGGGCTGTC AGCTGAAGTC CCTCCATTAT CTGGGATCTT 1700TGATGTCAGG GAAGGGGAGG CCTTGGTCTG AAGGGGCTGG AGTCAGGTCA 1750 GTAGAGGGAGGGTCTCAGGC CCTGCCAGGA GTGGACGTGA GGACCAAGCG 1800 GACTCGTCAC CCAGGACACCTGGACTCCAA TGAATTTGAC ATCTCTCGTT 1850 GTCCTTCGCG GAGGACCTGG TCACGTATGGCCAGATGTGG GTCCCCTCTA 1900 TCTCCTTCTG TACCATATCA GGGATGTGAG TTCTTGACATGAGAGATTCT 1950 CAAGCCAGCA AAAGGGTGGG ATTAGGCCCT ACAAGGAGAA AGGTGAGGGC2000 CCTGAGTGAG CACAGAGGGG ACCCTCCACC CAAGTAGAGT GGGGACCTCA 2050CGGAGTCTGG CCAACCCTGC TGAGACTTCT GGGAATCCGT GGCTGTGCTT 2100 GCAGTCTGCACACTGAAGGC CCGTGCATTC CTCTCCCAGG AATCAGGAGC 2150 TCCAGGAACC AGGCAGTGAGGCCTTGGTCT GAGTCAGTGC CTCAGGTCAC 2200 AGAGCAGAGG GGACGCAGAC AGTGCCAACACTGAAGGTTT GCCTGGAATG 2250 CACACCAAGG GCCCCACCCG CCCAGAACAA ATGGGACTCCAGAGGGCCTG 2300 GCCTCACCCT CCCTATTCTC AGTCCTGCAG CCTGAGCATG TGCTGGCCGG2350 CTGTACCCTG AGGTGCCCTC CCACTTCCTC CTTCAGGTTC TGAGGGGGAC 2400AGGCTGACAA GTAGGACCCG AGGCACTGGA GGAGCATTGA AGGAGAAGAT 2450 CTGTAAGTAAGCCTTTGTCA GAGCCTCCAA GGTTCAGTTC AGTTCTCACC 2500 TAAGGCCTCA CACACGCTCCTTCTCTCCCC AGGCCTGTGG GTCTTCATTG 2550 CCCAGCTCCT GCCCGCACTC CTGCCTGCTGCCCTGACCAG AGTCATC 2597 ATG CCT CTT GAG CAG AGG AGT CAG CAC TGC AAG CCTGAA GAA 2639 GGC CTT GAG GCC CGA GGA GAG GCC CTG GGC CTG GTG GGT GCG2681 CAG GCT CCT GCT ACT GAG GAG CAG CAG ACC GCT TCT TCC TCT 2723 TCTACT CTA GTG GAA GTT ACC CTG GGG GAG GTG CCT GCT GCC 2765 GAC TCA CCG AGTCCT CCC CAC AGT CCT CAG GGA GCC TCC AGC 2807 TTC TCG ACT ACC ATC AAC TACACT CTT TGG AGA CAA TCC GAT 2849 GAG GGC TCC AGC AAC CAA GAA GAG GAG GGGCCA AGA ATG TTT 2891 CCC GAC CTG GAG TCC GAG TTC CAA GCA GCA ATC AGT AGGAAG 2933 ATG GTT GAG TTG GTT CAT TTT CTG CTC CTC AAG TAT CGA GCC 2975AGG GAG CCG GTC ACA AAG GCA GAA ATG CTG GAG AGT GTC CTC 3017 AGA AAT TGCCAG GAC TTC TTT CCC GTG ATC TTC AGC AAA GCC 3059 TCC GAG TAC TTG CAG CTGGTC TTT GGC ATC GAG GTG GTG GAA 3101 GTG GTC CCC ATC AGC CAC TTG TAC ATCCTT GTC ACC TGC CTG 3143 GGC CTC TCC TAC GAT GGC CTG CTG GGC GAC AAT CAGGTC ATG 3185 CCC AAG ACA GGC CTC CTG ATA ATC GTC CTG GCC ATA ATC GCA3227 ATA GAG GGC GAC TGT GCC CCT GAG GAG AAA ATC TGG GAG GAG 3269 CTGAGT ATG TTG GAG GTG TTT GAG GGG AGG GAG GAC AGT GTC 3311 TTC GCA CAT CCCAGG AAG CTG CTC ATG CAA GAT CTG GTG CAG 3353 GAA AAC TAC CTG GAG TAC CGGCAG GTG CCC GGC AGT GAT CCT 3395 GCA TGC TAC GAG TTC CTG TGG GGT CCA AGGGCC CTC ATT GAA 3437 ACC AGC TAT GTG AAA GTC CTG CAC CAT ACA CTA AAG ATCGGT 3479 GGA GAA CCT CAC ATT TCC TAC CCA CCC CTG CAT GAA CGG GCT 3521TTG AGA GAG GGA GAA GAG TGA 3542 GTCTCAGCAC ATGTTGCAGC CAGGGCCAGTGGGAGGGGGT CTGGGCCAGT 3592 GCACCTTCCA GGGCCCCATC CATTAGCTTC CACTGCCTCGTGTGATATGA 3642 GGCCCATTCC TGCCTCTTTG AAGAGAGCAG TCAGCATTCT TAGCAGTGAG3692 TTTCTGTTCT GTTGGATGAC TTTGAGATTT ATCTTTCTTT CCTGTTGGAA 3742TTGTTCAAAT GTTCCTTTTA ACAAATGGTT GGATGAACTT CAGCATCCAA 3792 GTTTATGAATGACAGTAGTC ACACATAGTG CTGTTTATAT AGTTTAGGGG 3842 TAAGAGTCCT GTTTTTTATTCAGATTGGGA AATCCATTCC ATTTTGTGAG 3892 TTGTCACATA ATAACAGCAG TGGAATATGTATTTGCCTAT ATTGTGAACG 3942 AATTAGCAGT AAAATACATG ATACAAGGAA CTCAAAAGATAGTTAATTCT 3992 TGCCTTATAC CTCAGTCTAT TATGTAAAAT TAAAAATATG TGTATGTTTT4042 TGCTTCTTTG AGAATGCAAA AGAAATTAAA TCTGAATAAA TTCTTCCTGT 4092TCACTGGCTC ATTTCTTTAC CATTCACTCA GCATCTGCTC TGTGGAAGGC 4142 CCTGGTAGTAGTGGG 4157 662 base pairs nucleic acid single linear genomic DNA MAGE-21gene 10 GGATCCCCAT GGATCCAGGA AGAATCCAGT TCCACCCCTG CTGTGAACCC 50AGGGAAGTCA CGGGGCCGGA TGTGACGCCA CTGACTTGCG CGTTGGAGGT 100 CAGAGAACAGCGAGATTCTC GCCCTGAGCA ACGGCCTGAC GTCGGCGGAG 150 GGAAGCAGGC GCAGGCTCCGTGAGGAGGCA AGGTAAGATG CCGAGGGAGG 200 ACTGAGGCGG GCCTCACCCC AGACAGAGGGCCCCCAATAA TCCAGCGCTG 250 CCTCTGCTGC CAGGCCTGGA CCACCCTGCA GGGGAAGACTTCTCAGGCTC 300 AGTCGCCACC ACCTCACCCC GCCACCCCCC GCCGCTTTAA CCGCAGGGAA350 CTCTGGTGTA AGAGCTTTGT GTGACCAGGG CAGGGCTGGT TAGAAGTGCT 400CAGGGCCCAG ACTCAGCCAG GAATCAAGGT CAGGACCCCA AGAGGGGACT 450 GAGGGTAACCCCCCCGCACC CCCACCACCA TTCCCATCCC CCAACACCAA 500 CCCCACCCCC ATCCCCCAACACCAAACCCA CCACCATCGC TCAAACATCA 550 ACGGCACCCC CAAACCCCGA TTCCCATCCCCACCCATCCT GGCAGAATCG 600 GAGCTTTGCC CCTGCAATCA ACCCACGGAA GCTCCGGGAATGGCGGCCAA 650 GCACGCGGAT CC 662 1640 base pairs nucleic acid singlelinear cDNA to mRNA cDNA MAGE-3 11 GCCGCGAGGG AAGCCGGCCC AGGCTCGGTGAGGAGGCAAG GTTCTGAGGG 50 GACAGGCTGA CCTGGAGGAC CAGAGGCCCC CGGAGGAGCACTGAAGGAGA 100 AGATCTGCCA GTGGGTCTCC ATTGCCCAGC TCCTGCCCAC ACTCCCGCCT150 GTTGCCCTGA CCAGAGTCAT C 171 ATG CCT CTT GAG CAG AGG AGT CAG CAC TGCAAG CCT GAA GAA 213 GGC CTT GAG GCC CGA GGA GAG GCC CTG GGC CTG GTG GGTGCG 255 CAG GCT CCT GCT ACT GAG GAG CAG GAG GCT GCC TCC TCC TCT 297 TCTACT CTA GTT GAA GTC ACC CTG GGG GAG GTG CCT GCT GCC 339 GAG TCA CCA GATCCT CCC CAG AGT CCT CAG GGA GCC TCC AGC 381 CTC CCC ACT ACC ATG AAC TACCCT CTC TGG AGC CAA TCC TAT 423 GAG GAC TCC AGC AAC CAA GAA GAG GAG GGGCCA AGC ACC TTC 465 CCT GAC CTG GAG TCC GAG TTC CAA GCA GCA CTC AGT AGGAAG 507 GTG GCC GAG TTG GTT CAT TTT CTG CTC CTC AAG TAT CGA GCC 549 AGGGAG CCG GTC ACA AAG GCA GAA ATG CTG GGG AGT GTC GTC 591 GGA AAT TGG CAGTAT TTC TTT CCT GTG ATC TTC AGC AAA GCT 633 TCC AGT TCC TTG CAG CTG GTCTTT GGC ATC GAG CTG ATG GAA 675 GTG GAC CCC ATC GGC CAC TTG TAC ATC TTTGCC ACC TGC CTG 717 GGC CTC TCC TAC GAT GGC CTG CTG GGT GAC AAT CAG ATCATG 759 CCC AAG GCA GGC CTC CTG ATA ATC GTC CTG GCC ATA ATC GCA 801 AGAGAG GGC GAC TGT GCC CCT GAG GAG AAA ATC TGG GAG GAG 843 CTG AGT GTG TTAGAG GTG TTT GAG GGG AGG GAA GAC AGT ATG 885 TTG GGG GAT CCC AAG AAG CTGCTC ACC CAA CAT TTC GTG CAG 927 GAA AAC TAC CTG GAG TAC CGG CAG GTC CCCGGC AGT GAT CCT 969 GCA TGT TAT GAA TTC CTG TGG GGT CCA AGG GCC CTC GTTGAA 1011 ACC AGC TAT GTG AAA GTC CTG CAC CAT ATG GTA AAG ATC AGT 1053GGA GGA CCT CAC ATT TCC TAC CCA CCC CTG CAT GAG TGG GTT 1095 TTG AGA GAGGGG GAA GAG TGA 1116 GTCTGAGCAC GAGTTGCAGC CAGGGCCAGT GGGAGGGGGTCTGGGCCAGT 1166 GCACCTTCCG GGGCCGCATC CCTTAGTTTC CACTGCCTCC TGTGACGTGA1216 GGCCCATTCT TCACTCTTTG AAGCGAGCAG TCAGCATTCT TAGTAGTGGG 1266TTTCTGTTCT GTTGGATGAC TTTGAGATTA TTCTTTGTTT CCTGTTGGAG 1316 TTGTTCAAATGTTCCTTTTA ACGGATGGTT GAATGAGCGT CAGCATCCAG 1366 GTTTATGAAT GACAGTAGTCACACATAGTG CTGTTTATAT AGTTTAGGAG 1416 TAAGAGTCTT GttTTTTACT CAAATTgGGAAATCCATTCC ATTTTGTGAA 1466 TTGTGACATA ATAATAGCAG TGGTAAAAGT ATTTGCTTAAAATTGTGAGC 1516 GAATTAGCAA TAACATACAT GAGATAACTC AAGAAATCAA AAGATAGTTG1566 ATTCTTGCCT TGTACCTCAA TCTATTCTGT AAAATTAAAC AAATATGCAA 1616ACCAGGATTT CCTTGACTTC TTTG 1640 943 base pairs nucleic acid singlelinear genomic DNA MAGE-31 gene 12 GGATCCTCCA CCCCAGTAGA GTGGGGACCTCACAGAGTCT GGCCAACCCT 50 CCTGACAGTT CTGGGAATCC GTGGCTGCGT TTGCTGTCTGCACATTGGGG 100 GCCCGTGGAT TCCTCTCCCA GGAATCAGGA GCTCCAGGAA CAAGGCAGTG150 AGGACTTGGT CTGAGGCAGT GTCCTCAGGT CACAGAGTAG AGGGGgCTCA 200GATAGTGCCA ACGGTGAAGG TTTGCCTTGG ATTCAAACCA AGGGCCCCAC 250 CTGCCCCAGAACACATGGAC TCCAGAGCGC CTGGCCTCAC CCTCAATACT 300 TTCAGTCCTG CAGCCTCAGCATGCGCTGGC CGGATGTACC CTGAGGTGCC 350 CTCTCACTTC CTCCTTCAGG TTCTGAGGGGACAGGCTGAC CTGGAGGACC 400 AGAGGCCCCC GGAGGAGCAC TGAAGGAGAA GATCTGTAAGTAAGCCTTTG 450 TTAGAGCCTC CAAGGTTCCA TTCAGTACTC AGCTGAGGTC TCTCACATGC500 TCCCTCTCTC CCCAGGCCAG TGGGTCTCCA TTGCCCAGCT CCTGCCCACA 550CTCCCGCCTG TTGCCCTGAC CAGAGTCATC 580 ATG CCT CTT GAG CAG AGG AGT CAG CACTGC AAG CCT GAA GAA 622 GGC CTT GAG GCC CGA GGA GAg GCC CTG GGC CTG GTGGGT GCG 664 CAG GCT CCT GCT ACT GAG GAG CAG GAG GCT GCC TCC TCC TCT 706TCT AGT GTA GTT GAA GTC ACC CTG GGG GAG GTG CCT GCT GCC 748 GAG TCA CCAGAT CCT CCC CAG AGT CCT CAG GGA GCC TCC AGC 790 CTC CCC ACT ACC ATG AACTAC CCT CTC TGG AGC CAA TCC TAT 832 GAG GAC TCC AGC AAC CAA GAA GAG GAGGGG CCA AGC ACC TTC 874 CCT GAC CTG GAG TCT GAG TTC CAA GCA GCA CTC AGTAGG AAG 916 GTG GCC AAG TTG GTT CAT TTT CTG CTC 943 2531 base pairsnucleic acid single linear genomic DNA MAGE-4 gene 13 GGATCCAGGCCCTGCCTGGA GAAATGTGAG GGCCCTGAGT GAACACAGTG 50 GGGATCATCC ACTCCATGAGAGTGGGGACC TCACAGAGTC CAGCCTACCC 100 TCTTGATGGC ACTGAGGGAC CGGGGCTGTGCTTACAGTCT GCACCCTAAG 150 GGCCCATGGA TTCCTCTCCT AGGAGCTCCA GGAACAAGGCAGTGAGGCCT 200 TGGTCTGAGA CAGTGTCCTC AGGTTACAGA GCAGAGGATG CACAGGCTGT250 GCCAGCAGTG AATGTTTGCC CTGAATGCAC ACCAAGGGCC CCACCTGCCA 300CAAGACACAT AGGACTCCAA AGAGTCTGGC CTCACCTCCC TACCATCAAT 350 CCTGCAGAATCGACCTCTGC TGGCCGGCTA TACCCTGAGG TGCTCTCTCA 400 CTTCCTCCTT CAGGTTCTGAGCAGACAGGC CAACCGGAGA CAGGATTCCC 450 TGGAGGCCAC AGAGGAGCAC CAAGGAGAAGATCTGTAAGT AAGCCTTTGT 500 TAGAGCCTCT AAGATTTGGT TCTCAGCTGA GGTCTCTCACATGCTCCCTC 550 TCTCCGTAGG CCTGTGGGTC CCCATTGCCC AGCTTTTGCC TGCACTCTTG600 CCTGCTGCCC TGACCAGAGT CATC 624 ATG TCT TCT GAG CAG AAG AGT CAG CACTGC AAG CCT GAG GAA 666 GGC GTT GAG GCC CAA GAA GAG GCC CTG GGC CTG GTGGGT GCA 708 CAG GCT CCT ACT ACT GAG GAG CAG GAG GCT GCT GTC TCC TCC 750TCC TCT CCT CTG GTC CCT GGC ACC CTG GAG GAA GTG CCT GCT 792 GCT GAG TCAGCA GGT CCT CCC CAG AGT CCT CAG GGA GCC TCT 834 GCC TTA CCC ACT ACC ATCAGC TTC ACT TGC TGG AGG CAA CCC 876 AAT GAG GGT TCC AGC AGC CAA GAA GAGGAG GGG CCA AGC ACC 918 TCG CCT GAC GCA GAG TCC TTG TTC CGA GAA GCA CTCAGT AAC 960 AAG GTG GAT GAG TTG GCT CAT TTT CTG CTC CGC AAG TAT CGA 1002GCC AAG GAG CTG GTC ACA AAG GCA GAA ATG CTG GAG AGA GTC 1044 ATC AAA AATTAC AAG CGC TGC TTT CCT GTG ATC TTC GGC AAA 1086 GCC TCC GAG TCC CTG AAGATG ATC TTT GGC ATT GAC GTG AAG 1128 GAA GTG GAC CCC GCC AGC AAC ACC TACACC CTT GTC ACC TGC 1170 CTG GGC CTT TCC TAT GAT GGC CTG CTG GGT AAT AATCAG ATC 1212 TTT CCC AAG ACA GGC CTT CTG ATA ATC GTC CTG GGC ACA ATT1254 GCA ATG GAG GGC GAC AGC GCC TCT GAG GAG GAA ATC TGG GAG 1296 GAGCTG GGT GTG ATG GGG GTG TAT GAT GGG AGG GAG CAC ACT 1338 GTC TAT GGG GAGCCC AGG AAA CTG CTC ACC CAA GAT TGG GTG 1380 CAG GAA AAC TAC CTG GAG TACCGG CAG GTA CCC GGC AGT AAT 1422 CCT GCG CGC TAT GAG TTC CTG TGG GGT CCAAGG GCT CTG GCT 1464 GAA ACC AGC TAT GTG AAA GTC CTG GAG CAT GTG GTC AGGGTC 1506 AAT GCA AGA GTT CGC ATT GCC TAC CCA TCC CTG CGT GAA GCA 1548GCT TTG TTA GAG GAG GAA GAG GGA GTC TGA 1578 GCATGAGTTG CAGCCAGGGCTGTGGGGAAG GGGCAGGGCT GGGCCAGTGC 1628 ATCTAACAGC CCTGTGCAGC AGCTTCCCTTGCCTCGTGTA ACATGAGGCC 1678 CATTCTTCAC TCTGTTTGAA GAAAATAGTC AGTGTTCTTAGTAGTGGGTT 1728 TCTATTTTGT TGGATGACTT GGAGATTTAT CTCTGTTTCC TTTTACAATT1778 GTTGAAATGT TCCTTTTAAT GGATGGTTGA ATTAACTTCA GCATCCAAGT 1828TTATGAATCG TAGTTAACGT ATATTGCTGT TAATATAGTT TAGGAGTAAG 1878 AGTCTTGTTTTTTATTCAGA TTGGGAAATC CGTTCTATTT TGTGAATTTG 1928 GGACATAATA ACAGCAGTGGAGTAAGTATT TAGAAGTGTG AATTCACCGT 1978 GAAATAGGTG AGATAAATTA AAAGATACTTAATTCCCGCC TTATGCCTCA 2028 GTCTATTCTG TAAAATTTAA AAATATATAT GCATACCTGGATTTCCTTGG 2078 CTTCGTGAAT GTAAGAGAAA TTAAATCTGA ATAAATAATT CTTTCTGTTA2128 ACTGGCTCAT TTCTTCTCTA TGCACTGAGC ATCTGCTCTG TGGAAGGCCC 2178AGGATTAGTA GTGGAGATAC TAGGGTAAGC CAGACACACA CCTACCGATA 2228 GGGTATTAAGAGTCTAGGAG CGCGGTCATA TAATTAAGGT GACAAGATGT 2278 CCTCTAAGAT GTAGGGGAAAAGTAACGAGT GTGGGTATGG GGCTCCAGGT 2328 GAGAGTGGTC GGGTGTAAAT TCCCTGTGTGGGGCCTTTTG GGCTTTGGGA 2378 AACTGCATTT TCTTCTGAGG GATCTGATTC TAATGAAGCTTGGTGGGTCC 2428 AGGGCCAGAT TCTCAGAGGG AGAGGGAAAA GCCCAGATTG GAAAAGTTGC2478 TCTGAGCAGT TCCTTTGTGA CAATGGATGA ACAGAGAGGA GCCTCTACCT 2528 GGG2531 2531 base pairs nucleic acid single linear genomic DNA MAGE-41 gene14 GGATCCAGGC CCTGCCTGGA GAAATGTGAG GGCCCTGAGT GAACACAGTG 50 GGGATCATCCACTCCATGAG AGTGGGGACC TCACAGAGTC CAGCCTACCC 100 TCTTGATGGC ACTGAGGGACCGGGGCTGTG CTTACAGTCT GCACCCTAAG 150 GGCCCATGGA TTCCTCTCCT AGGAGCTCCAGGAACAAGGC AGTGAGGCCT 200 TGGTCTGAGA CAGTGTCCTC AGGTTACAGA GCAGAGGATGCACAGGCTGT 250 GCCAGCAGTG AATGTTTGCC CTGAATGCAC ACCAAGGGCC CCACCTGCCA300 CAAGACACAT AGGACTCCAA AGAGTCTGGC CTCACCTCCC TACCATCAAT 350CCTGCAGAAT CGACCTCTGC TGGCCGGCTA TACCCTGAGG TGCTCTCTCA 400 CTTCCTCCTTCAGGTTCTGA GCAGACAGGC CAACCGGAGA CAGGATTCCC 450 TGGAGGCCAC AGAGGAGCACCAAGGAGAAG ATCTGTAAGT AAGCCTTTGT 500 TAGAGCCTCT AAGATTTGGT TCTCAGCTGAGGTCTCTCAC ATGCTCCCTC 550 TCTCCGTAGG CCTGTGGGTC CCCATTGCCC AGCTTTTGCCTGCACTCTTG 600 CCTGCTGCCC TGAGCAGAGT CATC 624 ATG TCT TCT GAG CAG AAGAGT CAG CAC TGC AAG CCT GAG GAA 666 GGC GTT GAG GCC CAA GAA GAG GCC CTGGGC CTG GTG GGT GCG 708 CAG GCT CCT ACT ACT GAG GAG CAG GAG GCT GCT GTCTCC TCC 750 TCC TCT CCT CTG GTC CCT GGC ACC CTG GAG GAA GTG CCT GCT 792GCT GAG TCA GCA GGT CCT CCC CAG AGT CCT CAG GGA GCC TCT 834 GCC TTA CCCACT ACC ATC AGC TTC ACT TGC TGG AGG CAA CCC 876 AAT GAG GGT TCC AGC AGCCAA GAA GAG GAG GGG CCA AGC ACC 918 TCG CCT GAC GCA GAG TCC TTG TTC CGAGAA GCA CTC AGT AAC 960 AAG GTG GAT GAG TTG GCT CAT TTT CTG CTC CGC AAGTAT CGA 1002 GCC AAG GAG CTG GTC ACA AAG GCA GAA ATG CTG GAG AGA GTC1044 ATC AAA AAT TAC AAG CGC TGC TTT CCT GTG ATC TTC GGC AAA 1086 GCCTCC GAG TCC CTG AAG ATG ATC TTT GGC ATT GAC GTG AAG 1128 GAA GTG GAC CCCACC AGC AAC ACC TAC ACC CTT GTC ACC TGC 1170 CTG GGC CTT TCC TAT GAT GGCCTG CTG GGT AAT AAT CAG ATC 1212 TTT CCC AAG ACA GGC CTT CTG ATA ATC GTCCTG GGC ACA ATT 1254 GCA ATG GAG GGC GAC AGC GCC TCT GAG GAG GAA ATC TGGGAG 1296 GAG CTG GGT GTG ATG GGG GTG TAT GAT GGG AGG GAG CAC ACT 1338GTC TAT GGG GAG CCC AGG AAA CTG CTC ACC CAA GAT TGG GTG 1380 CAG GAA AACTAC CTG GAG TAC CGG CAG GTA CCC GGC AGT AAT 1422 CCT GCG CGC TAT GAG TTCCTG TGG GGT CCA AGG GCT CTG GCT 1464 GAA ACC AGC TAT GTG AAA GTC CTG GAGCAT GTG GTC AGG GTC 1506 AAT GCA AGA GTT CGC ATT GCC TAC CCA TCC CTG CGTGAA GCA 1548 GCT TTG TTA GAG GAG GAA GAG GGA GTC TGA 1578 GCATGAGTTGCAGCCAGGGC TGTGGGGAAG GGGCAGGGCT GGGCCAGTGC 1628 ATCTAACAGC CCTGTGCAGCAGCTTCCCTT GCCTCGTGTA ACATGAGGCC 1678 CATTCTTCAC TCTGTTTGAA GAAAATAGTCAGTGTTCTTA GTAGTGGGTT 1728 TCTATTTTGT TGGATGACTT GGAGATTTAT CTCTGTTTCCTTTTACAATT 1778 GTTGAAATGT TCCTTTTAAT GGATGGTTGA ATTAACTTCA GCATCCAAGT1828 TTATGAATCG TAGTTAACGT ATATTGCTGT TAATATAGTT TAGGAGTAAG 1878AGTCTTGTTT TTTATTCAGA TTGGGAAATC CGTTCTATTT TGTGAATTTG 1928 GGACATAATAACAGCAGTGG AGTAAGTATT TAGAAGTGTG AATTCACCGT 1978 GAAATAGGTG AGATAAATTAAAAGATACTT AATTCCCGCC TTATGCCTCA 2028 GTCTATTCTG TAAAATTTAA AAATATATATGCATACCTGG ATTTCCTTGG 2078 CTTCGTGAAT GTAAGAGAAA TTAAATCTGA ATAAATAATTCTTTCTGTTA 2128 ACTGGCTCAT TTCTTCTCTA TGCACTGAGC ATCTGCTCTG TGGAAGGCCC2178 AGGATTAGTA GTGGAGATAC TAGGGTAAGC CAGACACACA CCTACCGATA 2228GGGTATTAAG AGTCTAGGAG CGCGGTCATA TAATTAAGGT GACAAGATGT 2278 CCTCTAAGATGTAGGGGAAA AGTAACGAGT GTGGGTATGG GGCTCCAGGT 2328 GAGAGTGGTC GGGTGTAAATTCCCTGTGTG GGGCCTTTTG GGCTTTGGGA 2378 AACTCCATTT TCTTCTGAGG GATCTGATTCTAATGAAGCT TGGTGGGTCC 2428 AGGGCCAGAT TCTCAGAGGG AGAGGGAAAA GCCCAGATTGGAAAAGTTGC 2478 TCTGAGCGGT TCCTTTGTGA CAATGGATGA ACAGAGAGGA GCCTCTACCT2528 GGG 2531 1068 base pairs nucleic acid single linear cDNA to mRNAcDNA MAGE-4 15 G GGG CCA AGC ACC TCG CCT GAC GCA GAG TCC TTG TTC CGA 40GAA GCA CTC AGT AAC AAG GTG GAT GAG TTG GCT CAT TTT CTG 82 CTC CGC AAGTAT CGA GCC AAG GAG CTG GTC ACA AAG GCA GAA 124 ATG CTG GAG AGA GTC ATCAAA AAT TAC AAG CGC TGC TTT CCT 166 GTG ATC TTC GGC AAA GCC TCC GAG TCCCTG AAG ATG ATC TTT 208 GGC ATT GAC GTG AAG GAA GTG GAC CCC GCC AGC AACACC TAC 250 ACC CTT GTC ACC TGC CTG GGC CTT TCC TAT GAT GGC CTG CTG 292GGT AAT AAT CAG ATC TTT CCC AAG ACA GGC CTT CTG ATA ATC 334 GTC CTG GGCACA ATT GCA ATG GAG GGC GAC AGC GCC TCT GAG 376 GAG GAA ATC TGG GAG GAGCTG GGT GTG ATG GGG GTG TAT GAT 418 GGG AGG GAG CAC ACT GTC TAT GGG GAGCCC AGG AAA CTG CTC 460 ACC CAA GAT TGG GTG CAG GAA AAC TAC CTG GAG TACCGG CAG 502 GTA CCC GGC AGT AAT CCT GCG CGC TAT GAG TTC CTG TGG GGT 544CCA AGG GCT CTG GCT GAA ACC AGC TAT GTG AAA GTC CTG GAG 586 CAT GTG GTCAGG GTC AAT GCA AGA GTT CGC ATT GCC TAC CCA 628 TCC CTG CGT GAA GCA GCTTTG TTA GAG GAG GAA GAG GGA GTC 670 TGAGCATGAG TTGCAGCCAG GGCTGTGGGGAAGGGGCAGG GCTGGGCCAG 720 TGCATCTAAC AGCCCTGTGC AGCAGCTTCC CTTGCCTCGTGTAACATGAG 770 GCCCATTCTT CACTCTGTTT GAAGAAAATA GTCAGTGTTC TTAGTAGTGG820 GTTTCTATTT TGTTGGATGA CTTGGAGATT TATCTCTGTT TCCTTTTACA 870ATTGTTGAAA TGTTCCTTTT AATGGATGGT TGAATTAACT TCAGCATCCA 920 AGTTTATGAATCGTAGTTAA CGTATATTGC TGTTAATATA GTTTAGGAGT 970 AAGAGTCTTG TTTTTTATTCAGATTGGGAA ATCCGTTCTA TTTTGTGAAT 1020 TTGGGACATA ATAACAGCAG TGGAGTAAGTATTTAGAAGT GTGAATTC 1068 2226 base pairs nucleic acid single lineargenomic DNA MAGE-5 gene 16 GGATCCAGGC CTTGCCAGGA GAAAGGTGAG GGCCCTGTGTGAGCACAGAG 50 GGGACCATTC ACCCCAAGAG GGTGGAGACC TCACAGATTC CAGCCTACCC 100TCCTGTTAGC ACTGGGGGCC TGAGGCTGTG CTTGCAGTCT GCACCCTGAG 150 GGCCCATGCATTCCTCTTCC AGGAGCTCCA GGAAACAGAC ACTGAGGCCT 200 TGGTCTGAGG CCGTGCCCTCAGGTCACAGA GCAGAGGAGA TGCAGACGTC 250 TAGTGCCAGC AGTGAACGTT TGCCTTGAATGCACACTAAT GGCCCCCATC 300 GCCCCAGAAC ATATGGGACT CCAGAGCACC TGGCCTCACCCTCTCTACTG 350 TCAGTCCTGC AGAATCAGCC TCTGCTTGCT TGTGTACCCT GAGGTGCCCT400 CTCACTTTTT CCTTCAGGTT CTCAGGGGAC AGGCTGACCA GGATCACCAG 450GAAGCTCCAG AGGATCCCCA GGAGGCCCTA GAGGAGCACC AAAGGAGAAG 500 ATCTGTAAGTAAGCCTTTGT TAGAGCCTCC AAGGTTCAGT TTTTAGCTGA 550 GGCTTCTCAC ATGCTCCCTCTCTCTCCAGG CCAGTGGGTC TCCATTGCCC 600 AGCTCCTGCC CACACTCCTG CCTGTTGCGGTGACCAGAGT CGTC 644 ATG TCT CTT GAG CAG AAG AGT CAG CAC TGC AAG CCT GAGGAA 686 CTC CTC TGG TCC CAG GCA CCC TGG GGG AGG TGC CTG CTG CTG 728 GGTCAC CAG GTC CTC TCA AGA GTC CTC AGG GAG CCT CCG CCA 770 TCC CCA CTG CCATCG ATT TCA CTC TAT GGA GGC AAT CCA TTA 812 AGG GCT CCA GCA ACC AAG AAGAGG AGG GGC CAA GCA CCT CCC 854 CTG ACC CAG AGT CTG TGT TCC GAG CAG CACTCA GTA AGA AGG 896 TGG CTG ACT TGA 908 TTCATTTTCT GCTCCTCAAG TATTAAGTCAAGGAGCTGGT CACAAAGGCA 958 GAAATGCTGG AGAGCGTCAT CAAAAATTAC AAGCGCTGCTTTCCTGAGAT 1008 CTTCGGCAAA GCCTCCGAGT CCTTGCAGCT GGTCTTTGGC ATTGACGTGA1058 AGGAAGCGGA CCCCACCAGC AACACCTACA CCCTTGTCAC CTGCCTGGGA 1108CTCCTATGAT GGCCTGCTGG TTGATAATAA TCAGATCATG CCCAAGACGG 1158 GCCTCCTGATAATCGTCTTG GGCATGATTG CAATGGAGGG CAAATGCGTC 1208 CCTGAGGAGA AAATCTGGGAGGAGCTGAGT GTGATGAAGG TGTATGTTGG 1258 GAGGGAGCAC AGTGTCTGTG GGGAGCCCAGGAAGCTGCTC ACCCAAGATT 1308 TGGTGCAGGA AAACTACCTG GAGTACCGGC AGGTGCCCAGCAGTGATCCC 1358 ATATGCTATG AGTTACTGTG GGGTCCAAGG GCACTCGCTG CTTGAAAGTA1408 CTGGAGCACG TGGTCAGGGT CAATGCAAGA GTTCTCATTT CCTACCCATC 1458CCTGCGTGAA GCAGCTTTGA GAGAGGAGGA AGAGGGAGTC TGAGCATGAG 1508 CTGCAGCCAGGGCCACTGCG AGGGGGGCTG GGCCAGTGCA CCTTCCAGGG 1558 CTCCGTCCAG TAGTTTCCCCTGCCTTAATG TGACATGAGG CCCATTCTTC 1608 TCTCTTTGAA GAGAGCAGTC AACATTCTTAGTAGTGGGTT TCTGTTCTAT 1658 TGGATGACTT TGAGATTTGT CTTTGTTTCC TTTTGGAATTGTTCAAATGT 1708 TTCTTTTAAT GGGTGGTTGA ATGAACTTCA GCATTCAAAT TTATGAATGA1758 CAGTAGTCAC ACATAGTGCT GTTTATATAG TTTAGGAGTA AGAGTCTTGT 1808TTTTTATTCA GATTGGGAAA TCCATTCCAT TTTGTGAATT GGGACATAGT 1858 TACAGCAGTGGAATAAGTAT TCATTTAGAA ATGTGAATGA GCAGTAAAAC 1908 TGATGACATA AAGAAATTAAAAGATATTTA ATTCTTGCTT ATACTCAGTC 1958 TATTCGGTAA AATTTTTTTT AAAAAATGTGCATACCTGGA TTTCCTTGGC 2008 TTCTTTGAGA ATGTAAGACA AATTAAATCT GAATAAATCATTCTCCCTGT 2058 TCACTGGCTC ATTTATTCTC TATGCACTGA GCATTTGCTC TGTGGAAGGC2108 CCTGGGTTAA TAGTGGAGAT GCTAAGGTAA GCCAGACTCA CCCCTACCCA 2158CAGGGTAGTA AAGTCTAGGA GCAGCAGTCA TATAATTAAG GTGGAGAGAT 2208 GCCCTCTAAGATGTAGAG 2226 2305 base pairs nucleic acid single linear genomic DNAMAGE-51 gene 17 GGATCCAGGC CTTGCCAGGA GAAAGGTGAG GGCCCTGTGT GAGCACAGAG50 GGGACCATTC ACCCCAAGAG GGTGGAGACC TCACAGATTC CAGCCTACCC 100 TCCTGTTAGCACTGGGGGCC TGAGGCTGTG CTTGCAGTCT GCACCCTGAG 150 GGCCCATGCA TTCCTCTTCCAGGAGCTCCA GGAAACAGAC ACTGAGGCCT 200 TGGTCTGAGG CCGTGCCCTC AGGTCACAGAGCAGAGGAGA TGCAGACGTC 250 TAGTGCCAGC AGTGAACGTT TGCCTTGAAT GCACACTAATGGCCCCCATC 300 GCCCCAGAAC ATATGGGACT CCAGAGCACC TGGCCTCACC CTCTCTACTG350 TCAGTCCTGC AGAATCAGCC TCTGCTTGCT TGTGTACCCT GAGGTGCCCT 400CTCACTTTTT CCTTCAGGTT CTCAGGGGAC AGGCTGACCA GGATCACCAG 450 GAAGCTCCAGAGGATCCCCA GGAGGCCCTA GAGGAGCACC AAAGGAGAAG 500 ATCTGTAAGT AAGCCTTTGTTAGAGCCTCC AAGGTTCAGT TTTTAGCTGA 550 GGCTTCTCAC ATGCTCCCTC TCTCTCCAGGCCAGTGGGTC TCCATTGCCC 600 AGCTCCTGCC CACACTCCTG CCTGTTGCGG TGACCAGAGTCGTC 644 ATG TCT CTT GAG CAG AAG AGT CAG CAC TGC AAG CCT GAG GAA 686 GGCCTT GAC ACC CAA GAA GAG CCC TGG GCC TGG TGG GTG TGC 728 AGG CTG CCA CTACTG AGG AGC AGG AGG CTG TGT CCT CCT CCT 770 CTC CTC TGG TCC CAG GCA CCCTGG GGG AGG TGC CTG CTG CTG 812 GGT CAC CAG GTC CTC TCA AGA GTC CTC AGGGAG CCT CCG CCA 854 TCC CCA CTG CCA TCG ATT TCA CTC TAT GGA GGC AAT CCATTA 896 AGG GCT CCA GCA ACC AAG AAG AGG AGG GGC CAA GCA CCT CCC 938 CTGACC CAG AGT CTG TGT TCC GAG CAG CAC TCA GTA AGA AGG 980 TGG CTG ACT TGA992 TTCATTTTCT GCTCCTCAAG TATTAAGTCA AGGAGCCGGT CACAAAGGCA 1042GAAATGCTGG AGAGCGTCAT CAAAAATTAC AAGCGCTGCT TTCCTGAGAT 1092 CTTCGGCAAAGCCTCCGAGT CCTTGCAGCT GGTCTTTGGC ATTGACGTGA 1142 AGGAAGCGGA CCCCACCAGCAACACCTACA CCCTTGTCAC CTGCCTGGGA 1192 CTCCTATGAT GGCCTGGTGG TTTAATCAGATCATGCCCAA GACGGGCCTC 1242 CTGATAATCG TCTTGGGCAT GATTGCAATG GAGGGCAAATGCGTCCCTGA 1292 GGAGAAAATC TGGGAGGAGC TGGGTGTGAT GAAGGTGTAT GTTGGGAGGG1342 AGCACAGTGT CTGTGGGGAG CCCAGGAAGC TGCTCACCCA AGATTTGGTG 1392CAGGAAAACT ACCTGGAGTA CCGCAGGTGC CCAGCAGTGA TCCCATATGC 1442 TATGAGTTACTGTGGGGTCC AAGGGCACTC GCTGCTTGAA AGTACTGGAG 1492 CACGTGGTCA GGGTCAATGCAAGAGTTCTC ATTTCCTACC CATCCCTGCA 1542 TGAAGCAGCT TTGAGAGAGG AGGAAGAGGGAGTCTGAGCA TGAGCTGCAG 1592 CCAGGGCCAC TGCGAGGGGG GCTGGGCCAG TGCACCTTCCAGGGCTCCGT 1642 CCAGTAGTTT CCCCTGCCTT AATGTGACAT GAGGCCCATT CTTCTCTCTT1692 TGAAGAGAGC AGTCAACATT CTTAGTAGTG GGTTTCTGTT CTATTGGATG 1742ACTTTGAGAT TTGTCTTTGT TTCCTTTTGG AATTGTTCAA ATGTTCCTTT 1792 TAATGGGTGGTTGAATGAAC TTCAGCATTC AAATTTATGA ATGACAGTAG 1842 TCACACATAG TGCTGTTTATATAGTTTAGG AGTAAGAGTC TTGTTTTTTA 1892 TTCAGATTGG GAAATCCATT CCATTTTGTGAATTGGGACA TAGTTACAGC 1942 AGTGGAATAA GTATTCATTT AGAAATGTGA ATGAGCAGTAAAACTGATGA 1992 GATAAAGAAA TTAAAAGATA TTTAATTCTT GCCTTATACT CAGTCTATTC2042 GGTAAAATTT TTTTTTAAAA ATGTGCATAC CTGGATTTCC TTGGCTTCTT 2092TGAGAATGTA AGACAAATTA AATCTGAATA AATCATTCTC CCTGTTCACT 2142 GGCTCATTTATTCTCTATGC ACTGAGCATT TGCTCTGTGG AAGGCCCTGG 2192 GTTAATAGTG GAGATGCTAAGGTAAGCCAG ACTCACCCCT ACCCACAGGG 2242 TAGTAAAGTC TAGGAGCAGC AGTCATATAATTAAGGTGGA GAGATGCCCT 2292 CTAAGATGTA GAG 2305 225 base pairs nucleicacid single linear cDNA MAGE-6 gene 18 TAT TTC TTT CCT GTG ATC TTC AGCAAA GCT TCC GAT TCC TTG 42 CAG CTG GTC TTT GGC ATC GAG CTG ATG GAA GTGGAC CCC ATC 84 GGC CAC GTG TAC ATC TTT GCC ACC TGC CTG GGC CTC TCC TAC126 GAT GGC CTG CTG GGT GAC AAT CAG ATC ATG CCC AGG ACA GGC 168 TTC CTGATA ATC ATC CTG GCC ATA ATC GCA AGA GAG GGC GAC 210 TGT GCC CCT GAG GAG225 1947 base pairs nucleic acid single linear genomic DNA MAGE-7 gene19 TGAATGGACA ACAAGGGCCC CACACTCCCC AGAACACAAG GGACTCCAGA 50 GAGCCCAGCCTCACCTTCCC TACTGTCAGT CCTGCAGCCT CAGCCTCTGC 100 TGGCCGGCTG TACCCTGAGGTGCCCTCTCA CTTCCTCCTT CAGGTTCTCA 150 GCGGACAGGC CGGCCAGGAG GTCAGAAGCCCCAGGAGGCC CCAGAGGAGC 200 ACCGAAGGAG AAGATCTGTA AGTAGGCCTT TGTTAGGGCCTCCAGGGCGT 250 GGTTCACAAA TGAGGCCCCT CACAAGCTCC TTCTCTCCCC AGATCTGTGG300 GTTCCTCCCC ATCGCCCAGC TGCTGCCCGC ACTCCAGCCT GCTGCCCTGA 350CCAGAGTCAT CATGTCTTCT GAGCAGAGGA GTCAGCACTG CAAGCCTGAG 400 GATGCCTTGAGGCCCAAGGA CAGGAGGCTC TGGGCCTGGT GGGTGCGCAG 450 GCTCCCGCCA CCGAGGAGCACGAGGCTGCC TCCTCCTTCA CTCTGATTGA 500 AGGCACCCTG GAGGAGGTGC CTGCTGCTGGGTCCCCCAGT CCTCCCCTGA 550 GTCTCAGGGT TCCTCCTTTT CCCTGACCAT CAGCAACAACACTCTATGGA 600 GCCAATCCAG TGAGGGCACC AGCAGCCGGG AAGAGGAGGG GCCAACCACC650 TAGACACACC CCGCTCACCT GGCGTCCTTG TTCCA 685 ATG GGA AGG TGG CTG AGTTGG TTC GCT TCC TGC TGC ACA AGT 727 ATC GAG TCA AGG AGC TGG TCA CAA AGGCAG AAA TGC TGG ACA 769 GTG TCA TCA AAA ATT ACA AGC ACT AGT TTC CTT GTGATC TAT 811 GGC AAA GCC TCA GAG TGC ATG CAG GTG ATG TTT GGC ATT GAC 853ATG AAG GAA GTG GAC CCC GCG GCC ACT CCT ACG TCC TTG TCA 895 CCT GCT TGGGCC TCT CCT ACA ATG GCC TGC TGG GTG ATG ATC 937 AGA GCA TGC CCG AGA CCGGCC TTC TGA 964 TTATGGTCTT GACCATGATC TTAATGGAGG GCCACTGTGC CCCTGAGGAG1014 GCAATCTGGG AAGCGTTGAG TGTAATGGTG TATGATGGGA TGGAGCAGTT 1064TCTTTGGGCA GCTGAGGAAG CTGCTCACCC AAGATTGGGT GCAGGAAAAC 1114 TACCTGCAATACCGCCAGGT GCCCAGCAGT GATCCCCCGT GCTACCAGTT 1164 CCTGTGGGGT CCAAGGGCCCTCATTGAAAC CAGCTATGTG AAAGTCCTGG 1214 AGTATGCAGC CAGGGTCAGT ACTAAAGAGAGCATTTCCTA CCCATCCCTG 1264 CATGAAGAGG CTTTGGGAGA GGAGGAAGAG GGAGTCTGAGCAGAAGTTGC 1314 AGCCAGGGCC AGTGGGGCAG ATTGGGGGAG GGCCTGGGCA GTGCACGTTC1364 CACACATCCA CCACCTTCCC TGTCCTGTTA CATGAGGCCC ATTCTTCACT 1414CTGTGTTTGA AGAGAGCAGT CAATGTTCTC AGTAGCGGGG AGTGTGTTGG 1464 GTGTGAGGGAATACAAGGTG GACCATCTCT CAGTTCCTGT TCTCTTGGGC 1514 GATTTGGAGG TTTATCTTTGTTTCCTTTTG CAGTCGTTCA AATGTTCCTT 1564 TTAATGGATG GTGTAATGAA CTTCAACATTCATTTCATGT ATGACAGTAG 1614 GCAGACTTAC TGTTTTTTAT ATAGTTAAAA GTAAGTGCATTGTTTTTTAT 1664 TTATGTAAGA AAATCTATGT TATTTCTTGA ATTGGGACAA CATAACATAG1714 CAGAGGATTA AGTACCTTTT ATAATGTGAA AGAACAAAGC GGTAAAATGG 1764GTGAGATAAA GAAATAAAGA AATTAAATTG GCTGGGCACG GTGGCTCACG 1814 CCTGTAATCCCAGCACTTTA GGAGGCAGAG GCACGGGGAT CACGAGGTCA 1864 GGAGATCGAG ACCATTCTGGCTAACACAGT GAAACACCAT CTCTATTAAA 1914 AATACAAAAC TTAGCCGGGC GTGGTGGCGGGTG 1947 1810 base pairs nucleic acid single linear genomic DNA MAGE-8gene 20 GAGCTCCAGG AACCAGGCTG TGAGGTCTTG GTCTGAGGCA GTATCTTCAA 50TCACAGAGCA TAAGAGGCCC AGGCAGTAGT AGCAGTCAAG CTGAGGTGGT 100 GTTTCCCCTGTATGTATACC AGAGGCCCCT CTGGCATCAG AACAGCAGGA 150 ACCCCACAGT TCCTGGCCCTACCAGCCCTT TTGTCAGTCC TGGAGCCTTG 200 GCCTTTGCCA GGAGGCTGCA CCCTGAGATGCCCTCTCAAT TTCTCCTTCA 250 GGTTCGCAGA GAACAGGCCA GCCAGGAGGT CAGGAGGCCCCAGAGAAGCA 300 CTGAAGAAGA CCTGTAAGTA GACCTTTGTT AGGGCATCCA GGGTGTAGTA350 CCCAGCTGAG GCCTCTCACA CGCTTCCTCT CTCCCCAGGC CTGTGGGTCT 400CAATTGCCCA GCTCCGGCCC ACACTCTCCT GCTGCCCTGA CCTGAGTCAT 450 C 451 ATG CTTCTT GGG CAG AAG AGT CAG CGC TAC AAG GCT GAG GAA 493 GGC CTT CAG GCC CAAGGA GAG GCA CCA GGG CTT ATG GAT GTG 535 CAG ATT CCC ACA GCT GAG GAG CAGAAG GCT GCA TCC TCC TCC 577 TCT ACT CTG ATC ATG GGA ACC CTT GAG GAG GTGACT GAT TCT 619 GGG TCA CCA AGT CCT CCC CAG AGT CCT GAG GGT GCC TCC TCT661 TCC CTG ACT GTC ACC GAC AGC ACT CTG TGG AGC CAA TCC GAT 703 GAG GGTTCC AGC AGC AAT GAA GAG GAG GGG CCA AGC ACC TCC 745 CCG GAC CCA GCT CACCTG GAG TCC CTG TTC CGG GAA GCA CTT 787 GAT GAG AAA GTG GCT GAG TTA GTTCGT TTC CTG CTC CGC AAA 829 TAT CAA ATT AAG GAG CCG GTC ACA AAG GCA GAAATG CTT GAG 871 AGT GTC ATC AAA AAT TAC AAG AAC CAC TTT CCT GAT ATC TTC913 AGC AAA GCC TCT GAG TGC ATG CAG GTG ATC TTT GGC ATT GAT 955 GTG AAGGAA GTG GAC CCT GCC GGC CAC TCC TAC ATC CTT GTC 997 ACC TGC CTG GGC CTCTCC TAT GAT GGC CTG CTG GGT GAT GAT 1039 CAG AGT ACG CCC AAG ACC GGC CTCCTG ATA ATC GTC CTG GGC 1081 ATG ATC TTA ATG GAG GGC AGC CGC GCC CCG GAGGAG GCA ATC 1123 TGG GAA GCA TTG AGT GTG ATG GGG GCT GTA TGA 1156TGGGAGGGAG CACAGTGTCT ATTGGAAGCT CAGGAAGCTG CTCACCCAAG 1206 AGTGGGTGCAGGAGAACTAC CTGGAGTACC GCCAGGCGCC CGGCAGTGAT 1256 CCTGTGCGCT ACGAGTTCCTGTGGGGTCCA AGGGCCCTTG CTGAAACCAG 1306 CTATGTGAAA GTCCTGGAGC ATGTGGTCAGGGTCAATGCA AGAGTTCGCA 1356 TTTCCTACCC ATCCCTGCAT GAAGAGGCTT TGGGAGAGGAGAAAGGAGTT 1406 TGAGCAGGAG TTGCAGCTAG GGCCAGTGGG GCAGGTTGTG GGAGGGCCTG1456 GGCCAGTGCA CGTTCCAGGG CCACATCCAC CACTTTCCCT GCTCTGTTAC 1506ATGAGGCCCA TTCTTCACTC TGTGTTTGAA GAGAGCAGTC ACAGTTCTCA 1556 GTAGTGGGGAGCATGTTGGG TGTGAGGGAA CACAGTGTGG ACCATCTCTC 1606 AGTTCCTGTT CTATTGGGCGATTTGGAGGT TTATCTTTGT TTCCTTTTGG 1656 AATTGTTCCA ATGTTCCTTC TAATGGATGGTGTAATGAAC TTCAACATTC 1706 ATTTTATGTA TGACAGTAGA CAGACTTACT GCTTTTTATATAGTTTAGGA 1756 GTAAGAGTCT TGCTTTTCAT TTATACTGGG AAACCCATGT TATTTCTTGA1806 ATTC 1810 1412 base pairs nucleic acid single linear genomic DNAMAGE-9 gene 21 TCTGAGACAG TGTCCTCAGG TCGCAGAGCA GAGGAGACCC AGGCAGTGTC 50AGCAGTGAAG GTGAAGTGTT CACCCTGAAT GTGCACCAAG GGCCCCACCT 100 GCCCCAGCACACATGGGACC CCATAGCACC TGGCCCCATT CCCCCTACTG 150 TCACTCATAG AGCCTTGATCTCTGCAGGCT AGCTGCACGC TGAGTAGCCC 200 TCTCACTTCC TCCCTCAGGT TCTCGGGACAGGCTAACCAG GAGGACAGGA 250 GCCCCAAGAG GCCCCAGAGC AGCACTGACG AAGACCTGTAAGTCAGCCTT 300 TGTTAGAACC TCCAAGGTTC GGTTCTCAGC TGAAGTCTCT CACACACTCC350 CTCTCTCCCC AGGCCTGTGG GTCTCCATCG CCCAGCTCCT GCCCACGCTC 400CTGACTGCTG CCCTGACCAG AGTCATC 427 ATG TCT CTC GAG CAG AGG AGT CCG CACTGC AAG CCT GAT GAA 469 GAC CTT GAA GCC CAA GGA GAG GAC TTG GGC CTG ATGGGT GCA 511 CAG GAA CCC ACA GGC GAG GAG GAG GAG ACT ACC TCC TCC TCT 553GAC AGC AAG GAG GAG GAG GTG TCT GCT GCT GGG TCA TCA AGT 595 CCT CCC CAGAGT CCT CAG GGA GGC GCT TCC TCC TCC ATT TCC 637 GTC TAC TAC ACT TTA TGGAGC CAA TTC GAT GAG GGC TCC AGC 679 AGT CAA GAA GAG GAA GAG CCA AGC TCCTCG GTC GAC CCA GCT 721 CAG CTG GAG TTC ATG TTC CAA GAA GCA CTG AAA TTGAAG GTG 763 GCT GAG TTG GTT CAT TTC CTG CTC CAC AAA TAT CGA GTC AAG 805GAG CCG GTC ACA AAG GCA GAA ATG CTG GAG AGC GTC ATC AAA 847 AAT TAC AAGCGC TAC TTT CCT GTG ATC TTC GGC AAA GCC TCC 889 GAG TTC ATG CAG GTG ATCTTT GGC ACT GAT GTG AAG GAG GTG 931 GAC CCC GCC GGC CAC TCC TAC ATC CTTGTC ACT GCT CTT GGC 973 CTC TCG TGC GAT AGC ATG CTG GGT GAT GGT CAT AGCATG CCC 1015 AAG GCC GCC CTC CTG ATC ATT GTC CTG GGT GTG ATC CTA ACC1057 AAA GAC AAC TGC GCC CCT GAA GAG GTT ATC TGG GAA GCG TTG 1099 AGTGTG ATG GGG GTG TAT GTT GGG AAG GAG CAC ATG TTC TAC 1141 GGG GAG CCC AGGAAG CTG CTC ACC CAA GAT TGG GTG CAG GAA 1183 AAC TAC CTG GAG TAC CGG CAGGTG CCC GGC AGT GAT CCT GCG 1225 CAC TAC GAG TTC CTG TGG GGT TCC AAG GCCCAC GCT GAA ACC 1267 AGC TAT GAG AAG GTC ATA AAT TAT TTG GTC ATG CTC AATGCA 1309 AGA GAG CCC ATC TGC TAC CCA TCC CTT TAT GAA GAG GTT TTG 1351GGA GAG GAG CAA GAG GGA GTC TGA 1375 GCACCAGCCG CAGCCGGGGC CAAAGTTTGTGGGGTCA 1412 920 base pairs nucleic acid single linear genomic DNAMAGE-10 gene 22 ACCTGCTCCA GGACAAAGTG GACCCCACTG CATCAGCTCC ACCTACCCTA50 CTGTCAGTCC TGGAGCCTTG GCCTCTGCCG GCTGCATCCT GAGGAGCCAT 100 CTCTCACTTCCTTCTTCAGG TTCTCAGGGG ACAGGGAGAG CAAGAGGTCA 150 AGAGCTGTGG GACACCACAGAGCAGCACTG AAGGAGAAGA CCTGTAAGTT 200 GGCCTTTGTT AGAACCTCCA GGGTGTGGTTCTCAGCTGTG GCCACTTACA 250 CCCTCCCTCT CTCCCCAGGC CTGTGGGTCC CCATCGCCCAAGTCCTGCCC 300 ACACTCCCAC CTGCTACCCT GATCAGAGTC ATC 333 ATG CCT CGA GCTCCA AAG CGT CAG CGC TGC ATG CCT GAA GAA 375 GAT CTT CAA TCC CAA AGT GAGACA CAG GGC CTC GAG GGT GCA 417 CAG GCT CCC CTG GCT GTG GAG GAG GAT GCTTCA TCA TCC ACT 459 TCC ACC AGC TCC TCT TTT CCA TCC TCT TTT CCC TCC TCCTCC 501 TCT TCC TCC TCC TCC TCC TGC TAT CCT CTA ATA CCA AGC ACC 543 CCAGAG GAG GTT TCT GCT GAT GAT GAG ACA CCA AAT CCT CCC 585 CAG AGT GCT CAGATA GCC TGC TCC TCC CCC TCG GTC GTT GCT 627 TCC CTT CCA TTA GAT CAA TCTGAT GAG GGC TCC AGC AGC CAA 669 AAG GAG GAG AGT CCA AGC ACC CTA CAG GTCCTG CCA GAC AGT 711 GAG TCT TTA CCC AGA AGT GAG ATA GAT GAA AAG GTG ACTGAT 753 TTG GTG CAG TTT CTG CTC TTC AAG TAT CAA ATG AAG GAG CCG 795 ATCACA AAG GCA GAA ATA CTG GAG AGT GTC ATA AAA AAT TAT 837 GAA GAC CAC TTCCCT TTG TTG TTT AGT GAA GCC TCC GAG TGC 879 ATG CTG CTG GTC TTT GGC ATTGAT GTA AAG GAA GTG GAT CC 920 1107 base pairs nucleic acid singlelinear genomic DNA MAGE-11 gene 23 AGAGAACAGG CCAACCTGGA GGACAGGAGTCCCAGGAGAA CCCAGAGGAT 50 CACTGGAGGA GAACAAGTGT AAGTAGGCCT TTGTTAGATTCTCCATGGTT 100 CATATCTCAT CTGAGTCTGT TCTCACGCTC CCTCTCTCCC CAGGCTGTGG150 GGCCCCATCA CCCAGATATT TCCCACAGTT CGGCCTGCTG ACCTAACCAG 200AGTCATCATG CCTCTTGAGC AAAGAAGTCA GCACTGCAAG CCTGAGGAAG 250 CCTTCAGGCCCAAGAAGAAG ACCTGGGCCT GGTGGGTGCA CAGGCTCTCC 300 AAGCTGAGGA GCAGGAGGCTGCCTTCTTCT CCTCTACTCT GAATGTGGGC 350 ACTCTAGAGG AGTTGCCTGC TGCTGAGTCACCAAGTCCTC CCCAGAGTCC 400 TCAGGAAGAG TCCTTCTCTC CCACTGCCAT GGATGCCATCTTTGGGAGCC 450 TATCTGATGA GGGCTCTGGC AGCCAAGAAA AGGAGGGGCC AAGTACCTCG500 CCTGACCTGA TAGACCCTGA GTCCTTTTCC CAAGATATAC TACATGACAA 550GATAATTGAT TTGGTTCATT TATTCTCCGC AAGTATCGAG TCAAGGGGCT 600 GATCACAAAGGCAGAA 616 ATG CTG GGG AGT GTC ATC AAA AAT TAT GAG GAC TAC TTT CCT 658GAG ATA TTT AGG GAA GCC TCT GTA TGC ATG CAA CTG CTC TTT 700 GGC ATT GATGTG AAG GAA GTG GAC CCC ACT AGC CAC TCC TAT 742 GTC CTT GTC ACC TCC CTCAAC CTC TCT TAT GAT GGC ATA CAG 784 TGT AAT GAG CAG AGC ATG CCC AAG TCTGGC CTC CTG ATA ATA 826 GTC CTG GGT GTA ATC TTC ATG GAG GGG AAC TGC ATCCCT GAA 868 GAG GTT ATG TGG GAA GTC CTG AGC ATT ATG GGG GTG TAT GCT 910GGA AGG GAG CAC TTC CTC TTT GGG GAG CCC AAG AGG CTC CTT 952 ACC CAA AATTGG GTG CAG GAA AAG TAC CTG GTG TAC CGG CAG 994 GTG CCC GGC ACT GAT CCTGCA TGC TAT GAG TTC CTG TGG GGT 1036 CCA AGG GCC CAC GCT GAG ACC AGC AAGATG AAA GTT CTT GAG 1078 TAC ATA GCC AAT GCC AAT GGG AGG GAT CC 11072150 base pairs nucleic acid single linear genomic DNA smage-I 24TCTGTCTGCA TATGCCTCCA CTTGTGTGTA GCAGTCTCAA ATGGATCTCT 50 CTCTACAGACCTCTGTCTGT GTCTGGCACC CTAAGTGGCT TTGCATGGGC 100 ACAGGTTTCT GCCCCTGCATGGAGCTTAAA TAGATCTTTC TCCACAGGCC 150 TATACCCCTG CATTGTAAGT TTAAGTGGCTTTATGTGGAT ACAGGTCTCT 200 GCCCTTGTAT GCAGGCCTAA GTTTTTCTGT CTGCTTAACCCCTCCAAGTG 250 AAGCTAGTGA AAGATCTAAC CCACTTTTGG AAGTCTGAAA CTAGACTTTT300 ATGCAGTGGC CTAACAAGTT TTAATTTCTT CCACAGGGTT TGCAGAAAAG 350AGCTTGATCC ACGAGTTCAG AAGTCCTGGT ATGTTCCTAG AAAG 394 ATG TTC TCC TGG AAAGCT TCA AAA GCC AGG TCT CCA TTA AGT 436 CCA AGG TAT TCT CTA CCT GGT AGTACA GAG GTA CTT ACA GGT 478 TGT CAT TCT TAT CCT TCC AGA TTC CTG TCT GCCAGC TCT TTT 520 ACT TCA GCC CTG AGC ACA GTC AAC ATG CCT AGG GGT CAA AAG562 AGT AAG ACC CGC TCC CGT GCA AAA CGA CAG CAG TCA CGC AGG 604 GAG GTTCCA GTA GTT CAG CCC ACT GCA GAG GAA GCA GGG TCT 646 TCT CCT GTT GAC CAGAGT GCT GGG TCC AGC TTC CCT GGT GGT 688 TCT GCT CCT CAG GGT GTG AAA ACCCCT GGA TCT TTT GGT GCA 730 GGT GTA TCC TGC ACA GGC TCT GGT ATA GGT GGTAGA AAT GCT 772 GCT GTC CTG CCT GAT ACA AAA AGT TCA GAT GGC ACC CAG GCA814 GGG ACT TCC ATT CAG CAC ACA CTG AAA GAT CCT ATC ATG AGG 856 AAG GCTAGT GTG CTG ATA GAA TTC CTG CTA GAT AAA TTT AAG 898 ATG AAA GAA GCA GTTACA AGG AGT GAA ATG CTG GCA GTA GTT 940 AAC AAG AAG TAT AAG GAG CAA TTCCCT GAG ATC CTC AGG AGA 982 ACT TCT GCA CGC CTA GAA TTA GTC TTT GGT CTTGAG TTG AAG 1024 GAA ATT GAT CCC AGC ACT CAT TCC TAT TTG CTG GTA GGC AAA1066 CTG GGT CTT TCC ACT GAG GGA AGT TTG AGT AGT AAC TGG GGG 1108 TTGCCT AGG ACA GGT CTC CTA ATG TCT GTC CTA GGT GTG ATC 1150 TTC ATG AAG GGTAAC CGT GCC ACT GAG CAA GAG GTC TGG CAA 1192 TTT CTG CAT GGA GTG GGG GTATAT GCT GGG AAG AAG CAC TTG 1234 ATC TTT GGC GAG CCT GAG GAG TTT ATA AGAGAT GTA GTG CGG 1276 GAA AAT TAC CTG GAG TAC CGC CAG GTA CCT GGC AGT GATCCC 1318 CCA AGC TAT GAG TTC CTG TGG GGA CCC AGA GCC CAT GCT GAA 1360ACA ACC AAG ATG AAA GTC CTG GAA GTT TTA GCT AAA GTC AAT 1402 GGC ACA GTCCCT AGT GCC TTC CCT AAT CTC TAC CAG TTG GCT 1444 CTT AGA GAT CAG GCA GGAGGG GTG CCA AGA AGG AGA GTT CAA 1486 GGC AAG GGT GTT CAT TCC AAG GCC CCATCC CAA AAG TCC TCT 1528 AAC ATG TAG 1537 TTGAGTCTGT TCTGTTGTGTTTGAAAAACA GTCAGGCTCC TAATCAGTAG 1587 AGAGTTCATA GCCTACCAGA ACCAACATGCATCCATTCTT GGCCTGTTAT 1637 ACATTAGTAG AATGGAGGCT ATTTTTGTTA CTTTTCAAATGTTTGTTTAA 1687 CTAAACAGTG CTTTTTGCCA TGCTTCTTGT TAACTGCATA AAGAGGTAAC1737 TGTCACTTGT CAGATTAGGA CTTGTTTTGT TATTTGCAAC AAACTGGAAA 1787ACATTATTTT GTTTTTACTA AAACATTGTG TAACATTGCA TTGGAGAAGG 1837 GATTGTCATGGCAATGTGAT ATCATACAGT GGTGAAACAA CAGTGAAGTG 1887 GGAAAGTTTA TATTGTTAATTTTGAAAATT TTATGAGTGT GATTGCTGTA 1937 TACTTTTTTC TTTTTTGTAT AATGCTAAGTGAAATAAAGT TGGATTTGAT 1987 GACTTTACTC AAATTCATTA GAAAGTAAAT CGTAAAACTCTATTACTTTA 2037 TTATTTTCTT CAATTATGAA TTAAGCATTG GTTATCTGGA AGTTTCTCCA2087 GTAGCACAGG ATCTAGTATG AAATGTATCT AGTATAGGCA CTGACAGTGA 2137GTTATCAGAG TCT 2150 2099 base pairs nucleic acid single linear genomicDNA smage-II 25 ACCTTATTGG GTCTGTCTGC ATATGCCTCC ACTTGTGTGT AGCAGTCTCA50 AATGGATCTC TCTCTACAGA CCTCTGTCTG TGTCTGGCAC CCTAAGTGGC 100 TTTGCATGGGCACAGGTTTC TGCCCCTGCA TGGAGCTTAA ATAGATCTTT 150 CTCCACAGGC CTATACCCCTGCATTGTAAG TTTAAGTGGC TTTATGTGGA 200 TACAGGTCTC TGCCCTTGTA TGCAGGCCTAAGTTTTTCTG TCTGCTTAGC 250 CCCTCCAAGT GAAGCTAGTG AAAGATCTAA CCCACTTTTGGAAGTCTGAA 300 ACTAGACTTT TATGCAGTGG CCTAACAAGT TTTAATTTCT TCCACAGGGT350 TTGCAGAAAA GAGCTTGATC CACGAGTTCG GAAGTCCTGG TATGTTCCTA 400GAAAGATGTT CTCCTGGAAA GCTTCAAAAG CCAGGTCTCC ATTAAGTCCA 450 AGGTATTCTCTACCTGGTAG TACAGAGGTA CTTACAGGTT GTCATTCTTA 500 TCTTTCCAGA TTCCTGTCTGCCAGCTCTTT TACTTCAGCC CTGAGCACAG 550 TCAACATGCC TAGGGGTCAA AAGAGTAAGACCCGCTCCCG TGCAAAACGA 600 CAGCAGTCAC GCAGGGAGGT TCCAGTAGTT CAGCCCACTGCAGAGGAAGC 650 AGGGTCTTCT CCTGTTGACC AGAGTGCTGG GTCCAGCTTC CCTGGTGGTT700 CTGCTCCTCA GGGTGTGAAA ACCCCTGGAT CTTTTGGTGC AGGTGTATCC 750TGCACAGGCT CTGGTATAGG TGGTAGAAAT GCTGCTGTCC TGCCTGATAC 800 AAAAAGTTCAGATGGCACCC AGGCAGGGAC TTCCATTCAG CACACACTGA 850 AAGATCCTAT CATGAGGAAGGCTAGTGTGC TGATAGAATT CCTGCTAGAT 900 AAGTTTAAGA TGAAAGAAGC AGTTACAAGGAGTGAAATGC TGGCAGTAGT 950 TAACAAGAAG TATAAGGAGC AATTCCCTGA GATCCTCAGGAGAACTTCTG 1000 CACGCCTAGA ATTAGTCTTT GGTCTTGAGT TGAAGGAAAT TGATCCCAGC1050 ACTCATTCCT ATTTGCTGGT AGGCAAACTG GGTCTTTCCA CTGAGGGAAG 1100TTTGAGTAGT AACTGGGGGT TGCCTAGGAC AGGTCTCCTA ATGTCTGTCC 1150 TAGGTGTGATCTTCATGAAG GGTAACCGTG CCACTGAGCA AGAGGTCTGG 1200 CAATTTCTGC ATGGAGTGGGGGTATATGCT GGGAAGAAGC ACTTGATCTT 1250 TGGCGAGCCT GAGGAGTTTA TAAGAGATGTAGTGCGGGAA AATTACCTGG 1300 AGTACCGCCA GGTACCTGGC AGTGATCCCC CAAGCTATGAGTTCCTGTGG 1350 GGACCCAGAG CCCATGCTGA AACAACCAAG ATGAAAGTCC TGGAAGTTTT1400 AGCTAAAGTC AATGGCACAG TCCCTAGTGC CTTCCCTAAT CTCTACCAGT 1450TGGCTCTTAG AGATCAGGCA GGAGGGGTGC CAAGAAGGAG AGTTCAAGGC 1500 AAGGGTGTTCATTCCAAGGC CCCATCCCAA AAGTCCTCTA ACATGTAGTT 1550 GAGTCTGTTC TGTTGTGTTTGAAAAACAGT CAGGCTCCTA ATCAGTAGAG 1600 AGTTCATAGC CTACCAGAAC CAACATGCATCCATTCTTGG CCTGTTATAC 1650 ATTAGTAGAA TGGAGGCTAT TTTTGTTACT TTTCAAATGTTTGTTTAACT 1700 AAACAGTGCT TTTTGCCATG CTTCTTGTTA ACTGCATAAA GAGGTAACTG1750 TCACTTGTCA GATTAGGACT TGTTTTGTTA TTTGCAACAA ACTGGAAAAC 1800ATTATTTTGT TTTTACTAAA ACATTGTGTA ACATTGCATT GGAGAAGGGA 1850 TTGTCATGGCAATGTGATAT CATACAGTGG TGAAACAACA GTGAAGTGGG 1900 AAAGTTTATA TTGTTAGTTTTGAAAATTTT ATGAGTGTGA TTGCTGTATA 1950 CTTTTTTCTT TTTTGTATAA TGCTAAGTGAAATAAAGTTG GATTTGATGA 2000 CTTTACTCAA ATTCATTAGA AAGTAAATCA TAAAACTCTATTACTTTATT 2050 ATTTTCTTCA ATTATTAATT AAGCATTGGT TATCTGGAAG TTTCTCCAG2099 9 amino acids amino acids linear protein 26 Glu Ala Asp Pro Thr GlyHis Ser Tyr 5 20 nucleotides nucleic acid single linear 27 ACTCAGCTCCTCCCAGATTT 20 17 nucleotides nucleic acid single linear 28 GAAGAGGAGGGGCCAAG 17 18 nucleotides nucleic acid single linear 29 TCTTGTATCCTGGAGTCC 18 18 nucleotides nucleic acid single linear 30 TTGCCAAGATCTCAGGAA 18

We claim:
 1. A method for inducing an immune response in a patientsuffering from melanoma, colon cancer, non-small cell lung cancer,sarcoma or breast cancer, comprising administering an amount of MAGE-3protein to said patient sufficient to produce an immune responsetherein.
 2. The method of claim 1, wherein said patient suffers frommelanoma.
 3. The method of claim 1, wherein said patient suffers fromcolon cancer.
 4. The method of claim 1, wherein said patient suffersfrom non-small cell lung cancer.
 5. The method of claim 1, wherein saidpatient suffers from sarcoma.
 6. The method of claim 1, wherein saidpatient suffers from breast cancer.
 7. The method of claim 1, whereinsaid patient presents HLA-A1 molecules on surfaces of cells of saidmelanoma, colon cancer, non-small cell lung cancer, sarcoma or breastcancer.
 8. The method of claim 1, comprising administering said MAGE-3protein in a mixture with a pharmaceutically acceptable carrier whichforms a composition with said MAGE-3 protein.
 9. The method of claim 8,wherein said composition comprises an adjuvant.
 10. The method of claim8, wherein said composition is a vaccine.
 11. The method of claim 1,wherein said MAGE-3 protein is encoded by a nucleic acid moleculecomprising nucleotides 172-1116 of SEQ ID NO:
 11. 12. The method ofclaim 1, wherein said patient presents complexes of a tumor rejectionantigen consisting of an amino acid sequence found in MAGE-3 protein andHLA-A1 molecules on surges of cells of said melanoma, colon cancer,non-small cell lung cancer, sarcoma or breast cancer and said immuneresponse is proliferation of T cells specific for said cells ofmelanoma, colon cancer, non-small cell lung cancer, sarcoma or breastcancer.